Polynucleotides encoding antigenic HIV Type C polypeptides, polypeptides and uses thereof

ABSTRACT

The present invention relates to polynucleotides encoding immunogenic HIV type C Pol, Gag- and/or Env-containing polypeptides. Uses of the polynucleotides in applications including DNA immunization, generation of packaging cell lines, and production of Pol, Gag- and/or Env-containing proteins are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/475,704,filed Dec. 30, 1999 now abandoned, which in turn is related toprovisional patent applications Ser. Nos. 60/114,495, filed Dec. 31,1998 and 60/152,195, filed Sep. 1, 1999, from which priority is claimedunder 35 U.S.C. §119(e)(1) and which applications are incorporatedherein by reference in their entireties.

TECHNICAL FIELD

Polynucleotides encoding antigenic Type C HIV Gag-, Env- and/orPol-containing polypeptides are described, as are uses of thesepolynucleotides and polypeptide products in immunogenic compositions.Also described are polynucleotide sequences from South African variantsof HIV Type C.

BACKGROUND OF THE INVENTION

Acquired immune deficiency syndrome (AIDS) is recognized as one of thegreatest health threats facing modern medicine. There is, as yet, nocure for this disease. In 1983-1984, three groups independentlyidentified the suspected etiological agent of AIDS. See, e.g.,Barre-Sinoussi et al. (1983) Science 220:868-871; Montagnier et al., inHuman T-Cell Leukemia Viruses (Gallo, Essex & Gross, eds., 1984); Vilmeret al. (1984) The Lancet 1:753; Popovic et al. (1984) Science224:497-500; Levy et al. (1984) Science 225:840-842. These isolates werevariously called lymphadenopathy-associated virus (LAV), human T-celllymphotropic virus type III (HTLV-III), or AIDS-associated retrovirus(ARV). All of these isolates are strains of the same virus, and werelater collectively named Human Immunodeficiency Virus (HIV). With theisolation of a related AIDS-causing virus, the strains originally calledHIV are now termed HIV-1 and the related virus is called HIV-2 See,e.g., Guyader et al. (1987) Nature 326:662-669; Brun-Vezinet et al.(1986) Science 233:343-346; Clavel et al. (1986) Nature 324:691-695.

A great deal of information has been gathered about the HIV virus,however, to date an effective vaccine has not been identified. Severaltargets for vaccine development have been examined including the env andGag gene products encoded by HIV. Gag gene products include, but are notlimited to, Gag-polymerase and Gag-protease. Env gene products include,but are not limited to, monomeric gp120 polypeptides, oligomeric gp140polypeptides and gp160 polypeptides.

Haas, et al., (Current Biology 6(3):315-324, 1996) suggested thatselective codon usage by HIV-1 appeared to account for a substantialfraction of the inefficiency of viral protein synthesis. Andre, et al.,(J. Virol. 72(2):1497-1503, 1998) described an increased immune responseelicited by DNA vaccination employing a synthetic gp120 sequence withoptimized codon usage. Schneider, et al., (J. Virol. 71(7):4892-4903,1997) discuss inactivation of inhibitory (or instability) elements (INS)located within the coding sequences of the Gag and Gag-protease codingsequences.

The Gag proteins of HIV-1 are necessary for the assembly of virus-likeparticles. HIV-1 Gag proteins are involved in many stages of the lifecycle of the virus including, assembly, virion maturation after particlerelease, and early post-entry steps in virus replication. The roles ofHIV-1 Gag proteins are numerous and complex (Freed, E. O., Virology251:1-15, 1998).

Wolf, et al., (PCT International Application, WO 96/30523, published 3Oct. 1996; European Patent Application, Publication No. 0 449 116 A1,published 2 Oct. 1991) have described the use of altered pr55 Gag ofHIV-1 to act as a non-infectious retroviral-like particulate carrier, inparticular, for the presentation of immunologically important epitopes.Wang, et al., (Virology 200:524-534, 1994) describe a system to studyassembly of HIV Gag-β-galactosidase fusion proteins into virions. Theydescribe the construction of sequences encoding HIV Gag-β-galactosidasefusion proteins, the expression of such sequences in the presence of HIVGag proteins, and assembly of these proteins into virus particles.

Shiver, et al., (PCT International Application, WO 98/34640, published13 Aug. 1998) described altering HIV-1 (CAM1) Gag coding sequences toproduce synthetic DNA molecules encoding HIV Gag and modifications ofHIV Gag. The codons of the synthetic molecules were codons preferred bya projected host cell.

Recently, use of HIV Env polypeptides in immunogenic compositions hasbeen described. (see, U.S. Pat. No. 5,846,546 to Hurwitz et al., issuedDec. 8, 1998, describing immunogenic compositions comprising a mixtureof at least four different recombinant virus that each express adifferent HIV env variant; and U.S. Pat. No. 5,840,313 to Vahlne et al.,issued Nov. 24, 1998, describing peptides which correspond to epitopesof the HIV-1 gp120 protein). In addition, U.S. Pat. No. 5,876,731 to Siaet al, issued Mar. 2, 1999 describes candidate vaccines against HIVcomprising an amino acid sequence of a T-cell epitope of Gag linkeddirectly to an amino acid sequence of a B-cell epitope of the V3 loopprotein of an HIV-1 isolate containing the sequence GPGR. There remainsa need for antigenic HIV polypeptides, particularly Type C isolates.

SUMMARY OF THE INVENTION

The present invention relates to synthetic expression cassettes encodingHIV Type C Pol (e.g., p6pol, prot, p66RT, p15RNAseH, p31Int)-containingpolypeptides and to polynucleotides of novel HIV Type C variants. Inaddition, the present invention also relates to improved expression ofHIV Type C Pol- and/or Gag-containing polypeptides and production ofvirus-like particles, as well as, Env-containing polypeptides. Syntheticexpression cassettes encoding the HIV polypeptides (e.g., Gag-, pol-,prot-, reverse transcriptase, integrase and/or Env-containingpolypeptides) are described, as are uses of the expression cassettes.

One aspect of the present invention relates to expression cassettes andpolynucleotides contained therein. In one embodiment, an expressioncassette comprises a polynucleotide sequence encoding one or morePol-containing polypeptides, wherein the polynucleotide sequencecomprises a sequence having at least about 85%, preferably about 90%,more preferably about 95%, and more preferably about 98% sequence (andany integers between these values) identity to the sequences taught inthe present specification. The polynucleotide sequences encodingPol-containing polypeptides include, but are not limited to, those shownin SEQ ID NO:30, SEQ ID NO:31 and SEQ ID NO:32.

The polynucleotides encoding the Pol-containing polypeptides of thepresent invention may also include sequences encoding additionalpolypeptides. Such additional polynucleotides encoding polypeptides mayinclude, for example, coding sequences for other viral proteins (e.g.,hepatitis B or C or other HIV proteins, such as, polynucleotidesequences encoding an HIV Gag polypeptide, polynucleotide sequencesencoding an HIV Env polypeptide and/or polynucleotides encoding one ormore of vif, vpr, tat, rev, vpu and nef); cytokines or other transgenes.In one embodiment, the sequence encoding the HIV Pol polypeptide(s) canbe modified by deletions of coding regions corresponding to reversetranscriptase and integrase. Such deletions in the polymerasepolypeptide can also be made such that the polynucleotide sequencepreserves T-helper cell and CTL epitopes. Other antigens of interest maybe inserted into the polymerase as well.

In another embodiment, an expression cassette comprises a polynucleotidesequence encoding a polypeptide including an HIV Gag-containingpolypeptide, wherein the polynucleotide sequence encoding the Gagpolypeptide comprises a sequence having at least about 85%, preferablyabout 90%, more preferably about 95%, and most preferably about 98%sequence identity to the sequences taught in the present specification.The polynucleotide sequences encoding Gag-containing polypeptidesinclude, but are not limited to, the following polynucleotides:nucleotides 844-903 of FIG. 1 (a Gag major homology region) (SEQ IDNO:1); nucleotides 841-900 of FIG. 2 (a Gag major homology region) (SEQID NO:2); the sequence presented as FIG. 1 (SEQ ID NO:3); and thesequence presented as FIG. 2 (SEQ ID NO:4). As noted above, thepolynucleotides encoding the Gag-containing polypeptides of the presentinvention may also include sequences encoding additional polypeptides.

In another embodiment, an expression cassette comprises a polynucleotidesequence encoding a polypeptide including an HIV Env-containingpolypeptide, wherein the polynucleotide sequence encoding the Envpolypeptide comprises a sequence having at least about 85%, preferablyabout 90%, more preferably about 95%, and most preferably about 98%sequence identity to the sequences taught in the present specification.The polynucleotide sequences encoding Env-containing polypeptidesinclude, but are not limited to, the following polynucleotides:nucleotides 1213-1353 of FIG. 3 (SEQ ID NO:5) (an Env common region);nucleotides 82-1512 of FIG. 3 (SEQ ID NO:6) (a gp120 polypeptide);nucleotides 82-2025 of FIG. 3 (SEQ ID NO:7) (a gp140 polypeptide);nucleotides 82-2547 of FIG. 3 (SEQ ID NO:8) (a gp160 polypeptide);nucleotides 1-2547 of FIG. 3 (SEQ ID NO:9) (a gp160 polypeptide withsignal sequence); nucleotides 1513-2547 of FIG. 3 (SEQ ID NO:10) (a gp41polypeptide); nucleotides 1210-1353 of FIG. 4 (SEQ ID NO:11) (an Envcommon region); nucleotides 73-1509 of FIG. 4 (SEQ ID NO:12) (a gp120polypeptide); nucleotides 73-2022 of FIG. 4 (SEQ ID NO:13) (a gp140polypeptide); nucleotides 73-2565 of FIG. 4 (SEQ ID NO:14) (a gp160polypeptide); nucleotides 1-2565 of FIG. 4 (SEQ ID NO:15) (a gp160polypeptide with signal sequence); and nucleotides 1510-2565 of FIG. 4(SEQ ID NO:16) (a gp41 polypeptide).

The present invention further includes recombinant expression systemsfor use in selected host cells, wherein the recombinant expressionsystems employ one or more of the polynucleotides and expressioncassettes of the present invention. In such systems, the polynucleotidesequences are operably linked to control elements compatible withexpression in the selected host cell. Numerous expression controlelements are known to those in the art, including, but not limited to,the following: transcription promoters, transcription enhancer elements,transcription termination signals, polyadenylation sequences, sequencesfor optimization of initiation of translation, and translationtermination sequences. Exemplary transcription promoters include, butare not limited to those derived from CMV, CMV+intron A, SV40, RSV,HIV-Ltr, MMLV-ltr, and metallothionein.

In another aspect the invention includes cells comprising the expressioncassettes of the present invention where the polynucleotide sequence(e.g., encoding a Pol, Env- and/or Gag-containing polypeptide) isoperably linked to control elements compatible with expression in theselected cell. In one embodiment such cells are mammalian cells.Exemplary mammalian cells include, but are not limited to, BHK, VER0,HT1080, 293, RD, COS-7, and CHO cells. Other cells, cell types, tissuetypes, etc., that may be useful in the practice of the present inventioninclude, but are not limited to, those obtained from the following:insects (e.g., Trichoplusia ni (Tn5) and Sf9), bacteria, yeast, plants,antigen presenting cells (e.g., macrophage, monocytes, dendritic cells,B-cells, T-cells, stem cells, and progenitor cells thereof), primarycells, immortalized cells, tumor-derived cells.

In a further aspect, the present invention includes compositions forgenerating an immunological response, where the composition typicallycomprises at least one of the expression cassettes of the presentinvention and may, for example, contain combinations of expressioncassettes (such as one or more expression cassettes carrying aPol-polypeptide-encoding polynucleotide, one or more expressioncassettes carrying a Gag-polypeptide-encoding polynucleotide and/or oneor more expression cassettes carrying an Env-polypeptide-encodingpolynucleotide). Such compositions may further contain an adjuvant oradjuvants. The compositions may also contain one or more Pol-containingpolypeptides, one or more Gag-containing polypeptides and/or one or moreEnv-containing polypeptides. The Pol-containing polypetpides,Gag-containing polypeptides and/or Env-containing polypeptides maycorrespond to the polypeptides encoded by the expression cassette(s) inthe composition, or, the Pol-containing polypeptides, Gag-containingpolypeptides and/or Env-containing polypeptides may be different fromthose encoded by the expression cassettes. An example of thepolynucleotide in the expression cassette encoding the same polypeptideas is being provided in the composition is as follows: thepolynucleotide in the expression cassette encodes the Gag-polypeptide ofFIG. 1 (SEQ ID NO:3), and the polypeptide is the polypeptide encoded bythe sequence shown in FIG. 1 (SEQ ID NO:17). An example of thepolynucleotide in the expression cassette encoding a differentpolypeptide as is being provided in the composition is as follows: anexpression cassette having a polynucleotide encoding a Gag-polymerasepolypeptide, and the polypeptide provided in the composition may be aGag and/or Gag-protease polypeptide. In compositions containing bothexpression cassettes (or polynucleotides of the present invention) andpolypeptides, the Pol, Env and Gag expression cassettes of the presentinvention can be mixed and/or matched with Pol, Env-containing andGag-containing polypeptides described herein.

In another aspect the present invention includes methods of immunizationof a subject. In the method any of the above described compositions areinto the subject under conditions that are compatible with expression ofthe expression cassette in the subject. In one embodiment, theexpression cassettes (or polynucleotides of the present invention) canbe introduced using a gene delivery vector. The gene delivery vectorcan, for example, be a non-viral vector or a viral vector. Exemplaryviral vectors include, but are not limited to Sindbis-virus derivedvectors, retroviral vectors, and lentiviral vectors. Compositions usefulfor generating an immunological response can also be delivered using aparticulate carrier. Further, such compositions can be coated on, forexample, gold or tungsten particles and the coated particles deliveredto the subject using, for example, a gene gun. The compositions can alsobe formulated as liposomes. In one embodiment of this method, thesubject is a mammal and can, for example, be a human.

In a further aspect, the invention includes methods of generating animmune response in a subject, wherein the expression cassettes orpolynucleotides of the present invention are expressed in a suitablecell to provide for the expression of the Pol-, Env- and/orGag-containing polypeptides encoded by the polynucleotides of thepresent invention. The polypeptide(s) are then isolated (e.g.,substantially purified) and administered to the subject in an amountsufficient to elicit an immune response.

The invention further includes methods of generating an immune responsein a subject, where cells of a subject are transfected with any of theabove-described expression cassettes or polynucleotides of the presentinvention, under conditions that permit the expression of a selectedpolynucleotide and production of a polypeptide of interest (e.g.,encoded by any expression cassette of the present invention). By thismethod an immunological response to the polypeptide is elicited in thesubject. Transfection of the cells may be performed ex vivo and thetransfected cells are reintroduced into the subject. Alternately, or inaddition, the cells may be transfected in vivo in the subject. Theimmune response may be humoral and/or cell-mediated (cellular). In afurther embodiment, this method may also include administration of anEnv-, Pol- and/or Gag-containing polypeptide before, concurrently with,and/or after introduction of the expression cassette into the subject.

Further embodiments of the present invention include purifiedpolynucleotides. Exemplary polynucleotide sequences encodingGag-containing polypeptides include, but are not limited to, thefollowing polynucleotides: nucleotides 844-903 of FIG. 1 (SEQ ID NO:1)(a Gag major homology region); nucleotides 841-900 of FIG. 2 (SEQ IDNO:2) (a Gag major homology region); the sequence presented as FIG. 1(SEQ ID NO:3); and the sequence presented as FIG. 2 (SEQ ID NO:4).Exemplary polynucleotide sequences encoding Env-containing polypeptidesinclude, but are not limited to, the following polynucleotides:nucleotides 1213-1353 of FIG. 3 (SEQ ID NO:5) (an Env common region);nucleotides 82-1512 of FIG. 3 (SEQ ID NO:6) (a gp120 polypeptide);nucleotides 82-2025 of FIG. 3 (SEQ ID NO:7) (a gp140 polypeptide);nucleotides 82-2547 of FIG. 3 (SEQ ID NO:8) (a gp160 polypeptide);nucleotides 1-2547 of FIG. 3 (SEQ ID NO:9) (a gp160 polypeptide withsignal sequence); nucleotides 1513-2547 of FIG. 3 (SEQ ID NO:10) (a gp41polypeptide); nucleotides 1210-1353 of FIG. 4 (SEQ ID NO:11) (an Envcommon region); nucleotides 73-1509 of FIG. 4 (SEQ ID NO:12) (a gp120polypeptide); nucleotides 73-2022 of FIG. 4 (SEQ ID NO:13) (a gp140polypeptide); nucleotides 73-2565 of FIG. 4 (SEQ ID NO:14) (a gp160polypeptide); nucleotides 1-2565 of FIG. 4 (SEQ ID NO:15) (a gp160polypeptide with signal sequence); and nucleotides 1510-2565 of FIG. 4(SEQ ID NO:16) (a gp41 polypeptide). The polynucleotide sequenceencoding the Gag-containing and Env-containing polypeptides of thepresent invention typically have at least about 85%, preferably about90%, more preferably about 95%, and most preferably about 98% sequenceidentity to the sequences taught herein.

The polynucleotides of the present invention can be produced byrecombinant techniques, synthetic techniques, or combinations thereof.

Also described herein are novel Type C HIV sequences, for example,8_(—)5_ZA and 12_(—)5/1ZA and synthetic expression cassettes generatedfrom these sequences.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (SEQ ID NO:3) shows the nucleotide sequence of a polynucleotideencoding a synthetic Gag polypeptide. The nucleotide sequence shown wasobtained by modifying type C strain AF110965 and include furthermodifications of INS.

FIG. 2 (SEQ ID NO: 4) shows the nucleotide sequence of a polynucleotideencoding a synthetic Gag polypeptide. The nucleotide sequence shown wasobtained by modifying type C strain AF110967 and include furthermodifications of INS.

FIG. 3 (SEQ ID NO:9) shows the nucleotide sequence of a polynucleotideencoding a synthetic Env polypeptide. The nucleotide sequence depictsgp160 (including a signal peptide) and was obtained by modifying type Cstrain AF110968. The arrows indicate the positions of various regions ofthe polynucleotide, including the sequence encoding a signal peptide(nucleotides 1-81) (SEQ ID NO:18), a gp120 polypeptide (nucleotides82-1512) (SEQ ID NO:6), a gp41 polypeptide (nucleotides 1513-2547) (SEQID NO:10), a gp140 polypeptide (nucleotides 82-2025) (SEQ ID NO:7) and agp160 polypeptide (nucleotides 82-2547) (SEQ ID NO:8). The codonsencoding the signal peptide are modified (as described herein) from thenative HIV-1 signal sequence.

FIG. 4 (SEQ ID NO:15) shows the nucleotide sequence of a polynucleotideencoding a synthetic Env polypeptide. The nucleotide sequence depictsgp160 (including a signal peptide) and was obtained by modifying type Cstrain AF110975. The arrows indicate the positions of various regions ofthe polynucleotide, including the sequence encoding a signal peptide(nucleotides 1-72) (SEQ ID NO:19), a gp120 polypeptide (nucleotides73-1509) (SEQ ID NO:12), a gp41 polypeptide (nucleotides 1510-2565) (SEQID NO:16), a gp140 polypeptide (nucleotides 73-2022) (SEQ ID NO:13), anda gp160 polypeptide (nucleotides 73-2565) (SEQ ID NO:14). The codonsencoding the signal peptide are modified (as described herein) from thenative HIV-1 signal sequence.

FIG. 5 shows the location of some remaining INS in synthetic Gagsequences derived from AF110965. The changes made to these sequences areboxed in the Figures. The top line depicts a codon optimized sequence ofGag polypeptides from the indicated strains (SEQ ID NO:20). Thenucleotide(s) appearing below the line in the boxed region(s) depictschanges made to remove further INS and correspond to the sequencedepicted in FIG. 1 (SEQ ID NO:3).

FIG. 6 shows the location of some remaining INS in synthetic Gagsequences derived from AF110968. The changes made to these sequences areboxed in the Figures. The top line depicts a codon optimized sequence ofGag polypeptides from the indicated strains (SEQ ID NO:21). Thenucleotide(s) appearing below the line in the boxed region(s) depictschanges made to remove further INS and correspond to the sequencedepicted in FIG. 2 (SEQ ID NO:4).

FIG. 7 is a schematic depicting the selected domains in the Pol regionof HIV.

FIG. 8 (SEQ ID NO:30) depicts the nucleotide sequence of the constructdesignated PR975(+). “(+)” indicates that the reverse transcriptase isfunctional. This construct includes sequence from p2 (nucleotides 16 to54 of SEQ ID NO:30); p7 (nucleotides 55 to 219 of SEQ ID NO:30); p1/p6(nucleotides 220-375 of SEQ ID NO:30); prot (nucleotides 376 to 672 ofSEQ ID NO:30), reverse transcriptase (nucleotides 673 to 2352 of SEQ IDNO:30); and 6 amino acids of integrase shown in FIG. 7 (nucleotides 2353to 2370 of SEQ ID NO:30). In addition, the construct contains a multiplecloning site (MCS, nucleotides 2425 to 2463 of SEQ ID NO:30) forinsertion of a transgene and a YMDD epitope cassette (nucleotides 2371to 2424 of SEQ ID NO:30).

FIG. 9 (SEQ ID NO:31) depicts the nucleotide sequence of the constructdesignated PR975YM. As illustrated in FIG. 7, the RT region includes amutation in the catalytic center (mut. cat. center). “YM” refers toconstructs in which the nucleotides encode the amino acids AP instead ofYMDD in this region. Reverse transcriptase is not functional in thisconstruct. This construct includes sequence from the p2 (nucleotides 16to 54 of SEQ ID NO:31); p7 (nucleotides 55 to 219 of SEQ ID NO:31);p1/p6 (nucleotides 220 to 375 of SEQ ID NO:31); prot (nucleotides 376 to672 of SEQ ID NO:31); and reverse transcriptase (nucleotides 673 to 2346of SEQ ID NO:31) shown in FIG. 7, although the reverse transcriptaseprotein is not functional. In addition, the construct contains amultiple cloning site (MCS, nucleotides 2419 to 2457 of SEQ ID NO:31)for insertion of a transgene and a YMDD epitope cassette (nucleotides2365 to 2418 of SEQ ID NO:31).

FIG. 10 (SEQ ID NO:32) depicts the nucleotide sequence of the constructdesignated PR975YMWM. “YM” refers to constructs in which the nucleotidesencode the amino acids AP instead of YMDD in this region. “WM” refers toconstructs in which the nucleotides encode amino acids PI instead ofWMGY in this region. This construct includes sequence from the p2(nucleotides 16 to 54 of SEQ ID NO:32); p7 (nucleotides 55 to 219 of SEQID NO:32); p1/p6 (nucleotides 220 to 375 of SEQ ID NO:32); prot(nucleotides 376 to 672 of SEQ ID NO:32); and reverse transcriptase(nucleotides 673 to 2340 of SEQ ID NO:32) shown in FIG. 7, although thereverse transcriptase protein is not functional. In addition, theconstruct contains a multiple cloning site (MCS, nucleotides 2413 to2451 of SEQ ID NO:32) for insertion of a transgene and a YMDD epitopecassette (nucleotides 2359 to 2412 of SEQ ID NO:32).

FIG. 11 (SEQ ID NO:33) depicts the nucleotide sequence of 8_(—)5_ZA.Various regions are shown in Table B.

FIG. 12 (SEQ ID NO:34) depicts the wild type nucleotide sequence ofAF110975 Pol from p2gag until p7gag.

FIG. 13 (SEQ ID NO:35) depicts the wild type nucleotide sequence ofAF110975 Pol from p1 through the first 6 amino acids of the integraseprotein.

FIG. 14 (SEQ ID NO:36) depicts the nucleotide sequence of a cassetteencoding Ile178 through Serine 191 of reverse transcriptase.

FIG. 15 (SEQ ID NO:37) shows amino acid sequence which includes anepitope in the region of the catalytic center of the reversetranscriptase protein.

FIG. 16 (SEQ ID NO:45) depicts the nucleotide sequence of 12_(—)5/1ZA

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed.(Ausubel et al. eds., 1999, John Wiley & Sons); Molecular BiologyTechniques: An Intensive Laboratory Course, (Ream et al., eds., 1998,Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed.(Newton & Graham eds., 1997, Springer Verlag).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “an antigen”includes a mixture of two or more such agents.

1. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“Synthetic” sequences, as used herein, refers to Type C HIVpolypeptide-encoding polynucleotides whose expression has been optimizedas described herein, for example, by codon substitution and inactivationof inhibitory sequences. “Wild-type” or “native” sequences, as usedherein, refers to polypeptide encoding sequences that are essentially asthey are found in nature, e.g., Pol, Gag and/or Env encoding sequencesas found in Type C isolates, e.g., AF110965, AF110967, AF110968,AF110975 or 8_(—)5_ZA. The various regions of the HIV genome are shownin Table A, with numbering relative to 8_(—)5 ZA (SEQ ID NO:33). Thus,the term “Pol” refers to one or more of the following polypeptides:polymerase (p6Pol); protease (prot); reverse transcriptase (p66RT orRT); RNAseH (p15RNAseH); and/or integrase (p31Int or Int).

As used herein, the term “virus-like particle” or “VLP” refers to anonreplicating, viral shell, derived from any of several virusesdiscussed further below. VLPs are generally composed of one or moreviral proteins, such as, but not limited to those proteins referred toas capsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs can formspontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art and discussed more fully below. The presence of VLPsfollowing recombinant expression of viral proteins can be detected usingconventional techniques known in the art, such as by electronmicroscopy, X-ray crystallography, and the like. See, e.g., Baker etal., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol. (1994)68:4503-4505. For example, VLPs can be isolated by density gradientcentrifugation and/or identified by characteristic density banding.Alternatively, cryoelectron microscopy can be performed on vitrifiedaqueous samples of the VLP preparation in question, and images recordedunder appropriate exposure conditions.

By “particle-forming polypeptide” derived from a particular viralprotein is meant a full-length or near full-length viral protein, aswell as a fragment thereof, or a viral protein with internal deletions,which has the ability to form VLPs under conditions that favor VLPformation. Accordingly, the polypeptide may comprise the full-lengthsequence, fragments, truncated and partial sequences, as well as analogsand precursor forms of the reference molecule. The term thereforeintends deletions, additions and substitutions to the sequence, so longas the polypeptide retains the ability to form a VLP. Thus, the termincludes natural variations of the specified polypeptide sincevariations in coat proteins often occur between viral isolates. The termalso includes deletions, additions and substitutions that do notnaturally occur in the reference protein, so long as the protein retainsthe ability to form a VLP. Preferred substitutions are those which areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cystine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune system to make a humoral and/or cellular antigen-specificresponse. The term is used interchangeably with the term “immunogen.”Normally, a B-cell epitope will include at least about 5 amino acids butcan be as small as 3-4 amino acids. A T-cell epitope, such as a CTLepitope, will include at least about 7-9 amino acids, and a helperT-cell epitope at least about 12-20 amino acids. Normally, an epitopewill include between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term “antigen” denotes both subunit antigens, (i.e.,antigens which are separate and discrete from a whole organism withwhich the antigen is associated in nature), as well as, killed,attenuated or inactivated bacteria, viruses, fungi, parasites or othermicrobes. Antibodies such as anti-idiotype antibodies, or fragmentsthereof, and synthetic peptide mimotopes, which can mimic an antigen orantigenic determinant, are also captured under the definition of antigenas used herein. Similarly, an oligonucleotide or polynucleotide whichexpresses an antigen or antigenic determinant in vivo, such as in genetherapy and DNA immunization applications, is also included in thedefinition of antigen herein.

For purposes of the present invention, antigens can be derived from anyof several known viruses, bacteria, parasites and fungi, as describedmore fully below. The term also intends any of the various tumorantigens. Furthermore, for purposes of the present invention, an“antigen” refers to a protein which includes modifications, such asdeletions, additions and substitutions (generally conservative innature), to the native sequence, so long as the protein maintains theability to elicit an immunological response, as defined herein. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuringcell-mediated immune response include measurement of intracellularcytokines or cytokine secretion by T-cell populations, or by measurementof epitope specific T-cells (e.g., by the tetramer technique)(reviewedby McMichael, A. J., and O'Callaghan, C. A., J. Exp. Med.187(9)1367-1371, 1998; Mcheyzer-Williams, M. G., et al, Immunol. Rev.150:5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186:859-865, 1997).

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection to animmunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest. The immunogeniccomposition can be introduced directly into a recipient subject, such asby injection, inhalation, oral, intranasal and mucosal (e.g.,intra-rectally or intra-vaginally) administration.

By “subunit vaccine” is meant a vaccine composition which includes oneor more selected antigens but not all antigens, derived from orhomologous to, an antigen from a pathogen of interest such as from avirus, bacterium, parasite or fungus. Such a composition issubstantially free of intact pathogen cells or pathogenic particles, orthe lysate of such cells or particles. Thus, a “subunit vaccine” can beprepared from at least partially purified (preferably substantiallypurified) immunogenic polypeptides from the pathogen, or analogsthereof. The method of obtaining an antigen included in the subunitvaccine can thus include standard purification techniques, recombinantproduction, or synthetic production.

“Substantially purified” general refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA,genomic DNA sequences from viral or procaryotic DNA, and even syntheticDNA sequences. A transcription termination sequence may be located 3′ tothe coding sequence.

Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences.

A “nucleic acid” molecule can include, but is not limited to,procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA,genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “recombinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting procaryotic microorganisms or eucaryotic cell linescultured as unicellular entities, are used interchangeably, and refer tocells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Two or more polynucleotide sequences can be compared by determiningtheir “percent identity.” Two or more amino acid sequences likewise canbe compared by determining their “percent identity.” The percentidentity of two sequences, whether nucleic acid or peptide sequences, isgenerally described as the number of exact matches between two alignedsequences divided by the length of the shorter sequence and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981). This algorithm can be extended touse with peptide sequences using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An implementation of this algorithm for nucleic acid and peptidesequences is provided by the Genetics Computer Group (Madison, Wis.) intheir BestFit utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.). Other equally suitable programs for calculating thepercent identity or similarity between sequences are generally known inthe art.

For example, percent identity of a particular nucleotide sequence to areference sequence can be determined using the homology algorithm ofSmith and Waterman with a default scoring table and a gap penalty of sixnucleotide positions. Another method of establishing percent identity inthe context of the present invention is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages, theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated,the “Match” value reflects “sequence identity.” Other suitable programsfor calculating the percent identity or similarity between sequences aregenerally known in the art, such as the alignment program BLAST, whichcan also be used with default parameters. For example, BLASTN and BLASTPcan be used with the following default parameters: geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found on the internet.

One of skill in the art can readily determine the proper searchparameters to use for a given sequence in the above programs. Forexample, the search parameters may vary based on the size of thesequence in question. Thus, for example, a representative embodiment ofthe present invention would include an isolated polynucleotide having Xcontiguous nucleotides, wherein (i) the X contiguous nucleotides have atleast about 50% identity to Y contiguous nucleotides derived from any ofthe sequences described herein, (ii) X equals Y, and (iii) X is greaterthan or equal to 6 nucleotides and up to 5000 nucleotides, preferablygreater than or equal to 8 nucleotides and up to 5000 nucleotides, morepreferably 10-12 nucleotides and up to 5000 nucleotides, and even morepreferably 15-20 nucleotides, up to the number of nucleotides present inthe full-length sequences described herein (e.g., see the SequenceListing and claims), including all integer values falling within theabove-described ranges.

The synthetic expression cassettes (and purified polynucleotides) of thepresent invention include related polynucleotide sequences having about80% to 100%, greater than 80-85%, preferably greater than 90-92%, morepreferably greater than 95%, and most preferably greater than 98%sequence (including all integer values falling within these describedranges) identity to the synthetic expression cassette sequencesdisclosed herein (for example, to the claimed sequences or othersequences of the present invention) when the sequences of the presentinvention are used as the query sequence.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al., supraor Ausubel et al., supra). Such assays can be conducted using varyingdegrees of selectivity, for example, using conditions varying from lowto high stringency. If conditions of low stringency are employed, theabsence of non-specific binding can be assessed using a secondary probethat lacks even a partial degree of sequence identity (for example, aprobe having less than about 30% sequence identity with the targetmolecule), such that, in the absence of non-specific binding events, thesecondary probe will not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” typicallyhybridizes under conditions that allow detection of a target nucleicacid sequence of at least about 10-14 nucleotides in length having atleast approximately 70% sequence identity with the sequence of theselected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook, et al., supraor Ausubel et al., supra).

A first polynucleotide is “derived from” second polynucleotide if it hasthe same or substantially the same basepair sequence as a region of thesecond polynucleotide, its cDNA, complements thereof, or if it displayssequence identity as described above.

A first polypeptide is “derived from” a second polypeptide if it is (i)encoded by a first polynucleotide derived from a second polynucleotide,or (ii) displays sequence identity to the second polypeptides asdescribed above.

Generally, a viral polypeptide is “derived from” a particularpolypeptide of a virus (viral polypeptide) if it is (i) encoded by anopen reading frame of a polynucleotide of that virus (viralpolynucleotide), or (ii) displays sequence identity to polypeptides ofthat virus as described above.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences which areimmunologically identifiable with a polypeptide encoded by the sequence.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

By “nucleic acid immunization” is meant the introduction of a nucleicacid molecule encoding one or more selected antigens into a host cell,for the in vivo expression of an antigen, antigens, an epitope, orepitopes. The nucleic acid molecule can be introduced directly into arecipient subject, such as by injection, inhalation, oral, intranasaland mucosal administration, or the like, or can be introduced ex vivo,into cells which have been removed from the host. In the latter case,the transformed cells are reintroduced into the subject where an immuneresponse can be mounted against the antigen encoded by the nucleic acidmolecule.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting DNA of interest into a host cell. Such methods canresult in transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene delivery expression vectors include,but are not limited to, vectors derived from alphaviruses, pox virusesand vaccinia viruses. When used for immunization, such gene deliveryexpression vectors may be referred to as vaccines or vaccine vectors.

“T lymphocytes” or “T cells” are non-antibody producing lymphocytes thatconstitute a part of the cell-mediated arm of the immune system. T cellsarise from immature lymphocytes that migrate from the bone marrow to thethymus, where they undergo a maturation process under the direction ofthymic hormones. Here, the mature lymphocytes rapidly divide increasingto very large numbers. The maturing T cells become immunocompetent basedon their ability to recognize and bind a specific antigen. Activation ofimmunocompetent T cells is triggered when an antigen binds to thelymphocyte's surface receptors.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells. The term refers to both stableand transient uptake of the genetic material, and includes uptake ofpeptide- or antibody-linked DNAs.

A “vector” is capable of transferring gene sequences to target cells(e.g., viral vectors, non-viral vectors, particulate carriers, andliposomes). Typically, “vector construct,” “expression vector,” and“gene transfer vector,” mean any nucleic acid construct capable ofdirecting the expression of a gene of interest and which can transfergene sequences to target cells. Thus, the term includes cloning andexpression vehicles, as well as viral vectors.

Transfer of a “suicide gene” (e.g., a drug-susceptibility gene) to atarget cell renders the cell sensitive to compounds or compositions thatare relatively nontoxic to normal cells. Moolten, F. L. (1994) CancerGene Ther. 1:279-287. Examples of suicide genes are thymidine kinase ofherpes simplex virus (HSV-tk), cytochrome P450 (Manome et al. (1996)Gene Therapy 3:513-520), human deoxycytidine kinase (Manome et al.(1996) Nature Medicine 2(5):567-573) and the bacterial enzyme cytosinedeaminase (Dong et al. (1996) Human Gene Therapy 7:713-720). Cells whichexpress these genes are rendered sensitive to the effects of therelatively nontoxic prodrugs ganciclovir (HSV-tk), cyclophosphamide(cytochrome P450 2B1), cytosine arabinoside (human deoxycytidine kinase)or 5-fluorocytosine (bacterial cytosine deaminase). Culver et al. (1992)Science 256:1550-1552, Huber et al. (1994) Proc. Natl. Acad. Sci. USA91:8302-8306.

A “selectable marker” or “reporter marker” refers to a nucleotidesequence included in a gene transfer vector that has no therapeuticactivity, but rather is included to allow for simpler preparation,manufacturing, characterization or testing of the gene transfer vector.

A “specific binding agent” refers to a member of a specific binding pairof molecules wherein one of the molecules specifically binds to thesecond molecule through chemical and/or physical means. One example of aspecific binding agent is an antibody directed against a selectedantigen.

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The term does not denote a particular age. Thus,both adult and newborn individuals are intended to be covered. Thesystem described above is intended for use in any of the abovevertebrate species, since the immune systems of all of these vertebratesoperate similarly.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any undesirable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.2 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, “treatment” refers to any of (I) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

“Lentiviral vector”, and “recombinant lentiviral vector” refer to anucleic acid construct which carries, and within certain embodiments, iscapable of directing the expression of a nucleic acid molecule ofinterest. The lentiviral vector include at least one transcriptionalpromoter/enhancer or locus defining element(s), or other elements whichcontrol gene expression by other means such as alternate splicing,nuclear RNA export, post-translational modification of messenger, orpost-transcriptional modification of protein. Such vector constructsmust also include a packaging signal, long terminal repeats (LTRS) orportion thereof, and positive and negative strand primer binding sitesappropriate to the retrovirus used (if these are not already present inthe retroviral vector). Optionally, the recombinant lentiviral vectormay also include a signal which directs polyadenylation, selectablemarkers such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, aswell as one or more restriction sites and a translation terminationsequence. By way of example, such vectors typically include a 5′ LTR, atRNA binding site, a packaging signal, an origin of second strand DNAsynthesis, and a 3′LTR or a portion thereof.

“Lentiviral vector particle” as utilized within the present inventionrefers to a lentivirus which carries at least one gene of interest. Theretrovirus may also contain a selectable marker. The recombinantlentivirus is capable of reverse transcribing its genetic material (RNA)into DNA and incorporating this genetic material into a host cell's DNAupon infection. Lentiviral vector particles may have a lentiviralenvelope, a non-lentiviral envelope (e.g., an ampho or VSV-G envelope),or a chimeric envelope.

“Nucleic acid expression vector” or “Expression cassette” refers to anassembly which is capable of directing the expression of a sequence orgene of interest. The nucleic acid expression vector includes a promoterwhich is operably linked to the sequences or gene(s) of interest. Othercontrol elements may be present as well. Expression cassettes describedherein may be contained within a plasmid construct. In addition to thecomponents of the expression cassette, the plasmid construct may alsoinclude a bacterial origin of replication, one or more selectablemarkers, a signal which allows the plasmid construct to exist assingle-stranded DNA (e.g., a M13 origin of replication), a multiplecloning site, and a “mammalian” origin of replication (e.g., a SV40 oradenovirus origin of replication).

“Packaging cell” refers to a cell which contains those elementsnecessary for production of infectious recombinant retrovirus which arelacking in a recombinant retroviral vector. Typically, such packagingcells contain one or more expression cassettes which are capable ofexpressing proteins which encode Gag, pol and env proteins.

“Producer cell” or “vector producing cell” refers to a cell whichcontains all elements necessary for production of recombinant retroviralvector particles.

2. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

2.1. The HIV Genome

The HIV genome and various polypeptide-encoding regions are shown inTable A. The nucleotide positions are given relative to 8_(—)5_ZA (SEQID NO:33, FIG. 11). However, it will be readily apparent to one ofordinary skill in the art in view of the teachings of the presentdisclosure how to determine corresponding regions in other HIV strainsor variants (e.g., isolates HIV_(IIIb), HIV_(SF2), HIV-1_(SF162),HIV-1_(SF170), HIV_(LAV), HIV_(LAI), HIV_(MN), HIV-1_(CM235),HIV-1_(US4), other HIV-1 strains from diverse subtypes (e.g., subtypes,A through G, and O), HIV-2 strains and diverse subtypes (e.g.,HIV-2_(UC1) and HIV-2_(UC2)), and simian immunodeficiency virus (SIV).(See, e.g., Virology, 3rd Edition (W. K. Joklik ed. 1988); FundamentalVirology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991);Virology, 3rd Edition (Fields, B N, D M Knipe, P M Howley, Editors,1996, Lippincott-Raven, Philadelphia, Pa.; for a description of theseand other related viruses), using for example, sequence comparisonprograms (e.g., BLAST and others described herein) or identification andalignment of structural features (e.g., a program such as the “ALB”program described herein that can identify the various regions).

TABLE A Regions of the HIV Genome Region Position in nucleotide sequ.5'LTR  1-636 U3  1-457 R 458-553 U5 554-636 NFkB II 340-348 NFkB I354-362 Sp1 III 379-388 Sp1 II 390-398 Sp1 I 400-410 TATA Box 429-433TAR 474-499 Poly A signal 529-534 PBS 638-655 p7 binding region,packaging signal 685-791 Gag:  792-2285 p17  792-1178 p24 1179-1871Cyclophilin A bdg. 1395-1505 MHR 1632-1694 p2 1872-1907 P7 1908-2072Frameshift slip 2072-2078 p1 2073-2120 p6Gag 2121-2285 Zn-motif I1950-1991 Zn-motif II 2013-2054 Pol: 2072-5086 p6Pol 2072-2245 Prot2246-2542 p66RT 2543-4210 p15RNaseH 3857-4210 p31Int 4211-5086 Vif:5034-5612 Hydrophilic region 5292-5315 Vpr: 5552-5839 Oligomerization5552-5677 Amphipathic α-helix 5597-5653 Tat: 5823-6038 and 8417-8509Tat-1 exon 5823-6038 Tat-2 exon 8417-8509 N-terminal domain 5823-5885Trans-activation domain 5886-5933 Transduction domain 5961-5993 Rev:5962-6036 and 8416-8663 Rev-1 exon 5962-6036 Rev-2 exon 8416-8663High-affinity bdg. site 8439-8486 Leu-rich effector domain 8562-8588Vpu: 6060-6326 Transmembrane domain 6060-6161 Cytoplasmic domain6162-6326 Env (gp160): 6244-8853 Signal peptide 6244-6324 gp1206325-7794 V1 6628-6729 V2 6727-6852 V3 7150-7254 V4 7411-7506 V57663-7674 C1 6325-6627 C2 6853-7149 C3 7255-7410 C4 7507-7662 C57675-7794 CD4 binding 7540-7566 gp41 7795-8853 Fusion peptide 7789-7842Oligomerization domain 7924-7959 N-terminal heptad repeat 7921-8028C-terminal heptad repeat 8173-8280 Immunodominant region 8023-8076 Nef:8855-9478 Myristoylation 8858-8875 SH3 binding 9062-9091 Polypurinetract 9128-9154 SH3 binding 9296-9307

2.2 Synthetic Expression Cassettes

2.2.1 Modification of HIV-1-Type C Pol-, Prot-, Rt-, Int-, Gag and EnvNucleic Acid Coding Sequences

One aspect of the present invention is the generation of HIV-1 type CGag, Env and Pol coding sequences, and related sequences, havingimproved expression relative to the corresponding wild-type sequences.

2.2.1.1. Modification of Gag Nucleic Acid Coding Sequences

An exemplary embodiment of the present invention is illustrated hereinby modifying the Gag protein wild-type sequences obtained from theAF110965 and AF110967 strains of HIV-1, subtype C. (see, for example,Korber et al. (1998) Human Retroviruses and Aids, Los Alamos, N. Mex.Los Alamos National Laboratory; Novitsky et al. (1999) J. Virol.73(5):4427-4432, for molecular cloning of various subtype C clones fromBotswana). Gag sequence obtained from other Type C HIV-1 variants may bemanipulated in similar fashion following the teachings of the presentspecification. Such other variants include, but are not limited to, Gagprotein encoding sequences obtained from the isolates of HIV-1 Type C,for example as described in Novitsky et al., (1999), supra; Myers etal., infra; Virology, 3rd Edition (W. K. Joklik ed. 1988); FundamentalVirology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991);Virology, 3rd Edition (Fields, B N, D M Knipe, P M Howley, Editors,1996, Lippincott-Raven, Philadelphia, Pa. and on the World Wide Web(Internet).

First, the HIV-1 codon usage pattern was modified so that the resultingnucleic acid coding sequence was comparable to codon usage found inhighly expressed human genes (Example 1). The HIV codon usage reflects ahigh content of the nucleotides A or T of the codon-triplet. The effectof the HIV-1 codon usage is a high AT content in the DNA sequence thatresults in a decreased translation ability and instability of the mRNA.In comparison, highly expressed human codons prefer the nucleotides G orC. The Gag coding sequences were modified to be comparable to codonusage found in highly expressed human genes.

Second, there are inhibitory (or instability) elements (INS) locatedwithin the coding sequences of the Gag coding sequences. The RRE is asecondary RNA structure that interacts with the HIV encoded Rev-proteinto overcome the expression down-regulating effects of the INS. Toovercome the post-transcriptional activating mechanisms of RRE and Rev,the instability elements can be inactivated by introducing multiplepoint mutations that do not alter the reading frame of the encodedproteins. Subtype C Gag-encoding sequences having inactivated RRE sitesare shown in FIGS. 1 (SEQ ID NO:3), 2 (SEQ ID NO:4), 5 (SEQ ID NO:20)and 6 (SEQ ID NO:26).

Modification of the Gag polypeptide coding sequences results in improvedexpression relative to the wild-type coding sequences in a number ofmammalian cell lines (as well as other types of cell lines, including,but not limited to, insect cells). Further, expression of the sequencesresults in production of virus-like particles (VLPs) by these cell lines(see below).

2.2.1.2 Modification of Env Nucleic Acid Coding Sequences

Similarly, the present invention also includes modified Env proteins.Wild-type Env sequences are obtained from the AF110968 and AF110975strains of HIV-1, type C. (see, for example, Novitsky et al. (1999) J.Virol. 73(5):4427-4432, for molecular cloning of various subtype Cclones from Botswana). Env sequence obtained from other Type C HIV-1variants may be manipulated in similar fashion following the teachingsof the present specification. Such other variants include, but are notlimited to, Env protein encoding sequences obtained from the isolates ofHIV-1 Type C, described above.

The codon usage pattern for Env was modified as described above for Gagso that the resulting nucleic acid coding sequence was comparable tocodon usage found in highly expressed human genes. Experiments can beperformed in support of the present invention to show that the syntheticEnv sequences were capable of higher level of protein productionrelative to the native Env sequences.

Modification of the Env polypeptide coding sequences results in improvedexpression relative to the wild-type coding sequences in a number ofmammalian cell lines (as well as other types of cell lines, including,but not limited to, insect cells). Similar Env polypeptide codingsequences can be obtained, optimized and tested for improved expressionfrom a variety of isolates, including those described above for Gag.

2.2.1.3 Modification of Sequences Including Hiv-1 Pol Nucleic AcidCoding Sequences

The present invention also includes expression cassettes which includesynthetic Pol sequences. As noted above, “Pol” includes, but is notlimited to, the protein-encoding regions shown in FIG. 7, for examplepolymerase, protease, reverse transcriptase and/or integrase-containingsequences. The regions shown in FIG. 7 are described, for example, inWan et et al (1996) Biochem. J. 316:569-573; Kohl et al. (1988) PNAS USA85:4686-4690; Krausslich et al. (1988) J. Virol. 62:4393-4397; Coffin,“Retroviridae and their Replication” in Virology, pp 1437-1500 (Raven,New York, 1990); Patel et. al. (1995) Biochemistry 34:5351-5363. Thus,the synthetic expression cassettes exemplified herein include one ormore of these regions and one or more changes to the resulting aminoacid sequences.

Wild type Pol sequences were obtained from the AF110975 strains ofHIV-1, type C. (see, for example, Novitsky et al. (1999) J. Virol.73(5):4427-4432, for molecular cloning of various subtype C clones fromBotswana). SEQ ID NO:34 shows the wild type sequence from the p2 throughp7 region of Pol (see, FIG. 7 and Table A). SEQ ID NO:35 shows the wildtype sequence from p1 through the first 6 amino acids of integrase (see,FIG. 7 and Table A). Sequence obtained from other Type C HIV-1 variantsmay be manipulated in similar fashion following the teachings of thepresent specification. Such other variants include, but are not limitedto, Pol protein encoding sequences obtained from the isolates of HIV-1Type C described herein.

The codon usage pattern for Pol was modified as described above for Gagand Env so that the resulting nucleic acid coding sequence wascomparable to codon usage found in highly expressed human genes.

Table B shows the nucleotide positions of various regions found in thePol constructs exemplified herein (SEQ ID NOs: 30-32).

TABLE B Position in nucleotide sequence in construct PR975(+) PR975YMPR975(+) YMWM Region Seq Id No: 30 Seq Id No: 31 Seq Id No: 32 Sal 1restriction site 1-6 1-6 1-6 Kozak start codon  7-16  7-16  7-16 p216-54 16-54 16-54 P7  55-219  55-219  55-219 p1/p6 pol 220-375 220-375220-375 Insertion mutation 225 225 225 for in frame p10Protease 376-672376-672 376-672 p66RT  673-2352  673-2346  673-2340 p51RT  673-1992 673-1986  673-1980 p15RNaseH 1993-2352 1993-2346 1993-2340 catalyticcenter 1219-1230 1219-1224 1219-1224 region (YMDD) primer grip region1357-1368 1351-1362 1351-1356 (WMGy) 6aa Integrase 2353-2370 2347-23642341-2358 YMDD epitope 2371-2424 2365-2418 2359-2412 cassette (incl.5′ + 3′ Gly) MCS (multiple 2425-2463 2419-2457 2413-2451 cloning site)EcoR 1 restriction 2464-2469 2458-2463 2452-2457 site

As shown in Table B, exemplary constructs were modified in various ways.For example, the expression constructs exemplified herein includesequence that encodes the first 6 amino acids of the integrasepolypeptide. This 6 amino acid region is believed to provide a cleavagerecognition site recognized by HIV protease (see, e.g., McCornack et al.(1997) FEBS Letts 414:84-88). As noted above, certain constructsexemplified herein include a multiple cloning site (MCS) for insertionof one or more transgenes, typically at the 3′ end of the construct. Inaddition, a cassette encoding a catalytic center epitope derived fromthe catalytic center in RT is typically included 3′ of the sequenceencoding 6 amino acids of integrase. This cassette (SEQ ID NO:36)encodes Ile178 through Serine 191 of RT (amino acids 3 through 16 of SEQID NO:37) and was added to keep this well conserved region as a possibleCTL epitope. Further, the constructs contain an insertion mutations(position 225 of SEQ ID NOs:30 to 32) to preserve the reading frame.(see, e.g., Park et al. (1991) J. Virol. 65:5111).

In certain embodiments, the catalytic center and/or primer grip regionof RT are modified. The catalytic center and primer grip regions of RTare described, for example, in Patel et al. (1995) Biochem. 34:5351 andPalaniappan et al. (1997) J. Biol. Chem. 272(17):11157. For example, inthe construct designated PR975YM (SEQ ID NO:31), wild type sequenceencoding the amino acids YMDD at positions 183-185 of p66 RT, numberedrelative to AF110975, are replaced with sequence encoding the aminoacids “AP”. In the construct designated PR975YMWM (SEQ ID NO:32), thesame mutation in YMDD is made and, in addition, the primer grip region(amino acids WMGY, residues 229-232 of p66RT, numbered relative toAF110975) are replaced with sequence encoding the amino acids “PI.”

For the Pol sequence, the changes in codon usage are typicallyrestricted to the regions up to the −1 frameshift and starting again atthe end of the Gag reading frame; however, regions within the frameshifttranslation region can be modified as well. Finally, inhibitory (orinstability) elements (INS) located within the coding sequences of theprotease polypeptide coding sequence can be altered as well.

Experiments can be performed in support of the present invention to showthat the synthetic Pol sequences were capable of higher level of proteinproduction relative to the native Pol sequences. Modification of the Polpolypeptide coding sequences results in improved expression relative tothe wild-type coding sequences in a number of mammalian cell lines (aswell as other types of cell lines, including, but not limited to, insectcells). Similar Pol polypeptide coding sequences can be obtained,optimized and tested for improved expression from a variety of isolates,including those described above for Gag.

2.2.1.4 Modification of Sequences from 8_(—)5_ZA

The present invention also includes expression cassettes which includesynthetic HIV Type C sequences derived from 8_(—)5_ZA (SEQ ID NO:33).Wild-type sequences for various polypeptide-encoding regions areobtained from #8_(—)5_ZA (SEQ ID NO:33) and manipulated in similarfashion following the teachings of the present specification. The codonusage pattern for 8_(—)5_ZA is modified as described above for Gag, Envand Pol so that the resulting nucleic acid coding sequence is comparableto codon usage found in highly expressed human genes. Experiments can beperformed in support of the present invention to show that the synthetic8_(—)5_ZA sequences were capable of higher level of protein productionrelative to the native 8_(—)5_ZA sequences.

Modification of the 8_(—) 5 ZA polypeptide coding sequences results inimproved expression relative to the wild-type coding sequences in anumber of mammalian cell lines (as well as other types of cell lines,including, but not limited to, insect cells).

2.2.1.5 Further Modification of Sequences Including HIV-1 Nucleic AcidCoding Sequences

The Type C HIV polypeptide-encoding expression cassettes describedherein may also contain one or more further sequences encoding, forexample, one or more transgenes. Further sequences (e.g., transgenes)useful in the practice of the present invention include, but are notlimited to, further sequences are those encoding further viralepitopes/antigens {including but not limited to, HCV antigens (e.g., E1,E2; Houghton, M., et al., U.S. Pat. No. 5,714,596, issued Feb. 3, 1998;Houghton, M., et al., U.S. Pat. No. 5,712,088, issued Jan. 27, 1998;Houghton, M., et al., U.S. Pat. No. 5,683,864, issued Nov. 4, 1997;Weiner, A. J., et al., U.S. Pat. No. 5,728,520, issued Mar. 17, 1998;Weiner, A. J., et al., U.S. Pat. No. 5,766,845, issued Jun. 16, 1998;Weiner, A. J., et al., U.S. Pat. No. 5,670,152, issued Sep. 23, 1997;all herein incorporated by reference), HIV antigens (e.g., derived fromtat, rev, nef and/or env); and sequences encoding tumorantigens/epitopes. Further sequences may also be derived from non-viralsources, for instance, sequences encoding cytokines such interleukin-2(IL-2), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6(IL-6), interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colonystimulating factor (GM-CSF), interleukin-1 alpha (IL-11), interleukin-11(IL-11), MIP-11, tumor necrosis factor (TNF), leukemia inhibitory factor(LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand, commerciallyavailable from several vendors such as, for example, Genzyme(Framingham, Mass.), Genentech (South San Francisco, Calif.), Amgen(Thousand Oaks, Calif.), R&D Systems and Immunex (Seattle, Wash.).Additional sequences are described below, for example in Section 2.3.Also, variations on the orientation of the Gag and other codingsequences, relative to each other, are described below.

Gag, Env, and Pol polypeptide coding sequences can be obtained fromother Type C HIV isolates, see, e.g., Myers et al. Los Alamos Database,Los Alamos National Laboratory, Los Alamos, N. Mex. (1992); Myers etal., Human Retroviruses and Aids, 1997, Los Alamos, N. Mex.: Los AlamosNational Laboratory. Synthetic expression cassettes can be generatedusing such coding sequences as starting material by following theteachings of the present specification (e.g., see Example 1).

Further, the synthetic expression cassettes of the present inventioninclude related Pol, Gag and/or containing polypeptide sequences havinggreater than 85%, preferably greater than 90%, more preferably greaterthan 95%, and most preferably greater than 98% sequence identity to thesynthetic expression cassette sequences disclosed herein (for example,(SEQ ID NOs:30-32; SEQ ID NOs: 3, 4, 20, and 21 and SEQ ID NOs:5-17).Various coding regions are indicated in FIGS. 3 and 4, for example inFIG. 3 (AF110968), nucleotides 1-81 (SEQ ID NO:18) encode a signalpeptide, nucleotides 82-1512 (SEQ ID NO:6) encode a gp120 polypeptide,nucleotides 1513 to 2547 (SEQ ID NO:10) encode a gp41 polypeptide,nucleotides 82-2025 (SEQ ID NO:7) encode a gp140 polypeptide andnucleotides 82-2547 (SEQ ID NO:8) encode a gp160 polypeptide.

2.2.3 Expression of Synthetic Sequences Encoding HIV-1 Pol, Gag or Envand Related Polypeptides

Synthetic Pol-, Gag- and/or Env-encoding sequences (expressioncassettes) of the present invention can be cloned into a number ofdifferent expression vectors to evaluate levels of expression and, inthe case of Gag, production of VLPs. The synthetic DNA fragments forPol, Env and Gag can be cloned into eucaryotic expression vectors,including, a transient expression vector, CMV-promoter-based mammalianvectors, and a shuttle vector for use in baculovirus expression systems.Corresponding wild-type sequences can also be cloned into the samevectors.

These vectors can then be transfected into a several different celltypes, including a variety of mammalian cell lines (293, RD, COS-7, andCHO, cell lines available, for example, from the A.T.C.C.). The celllines are then cultured under appropriate conditions and the levels ofp24 (Gag) or, gp160 or gp120 (Env) expression in supernatants can beevaluated (Example 2). Env polypeptides include, but are not limited to,for example, native gp160, oligomeric gp140, monomeric gp120 as well asmodified sequences of these polypeptides. The results of these assaysdemonstrate that expression of synthetic Pol, Env, Gag encodingsequences are significantly higher than corresponding wild-typesequences.

Further, Western Blot analysis can be used to show that cells containingthe synthetic Pol, Gag or Env expression cassette produce the expectedprotein at higher per-cell concentrations than cells containing thenative expression cassette. The Pol, Gag and Env proteins can be seen inboth cell lysates and supernatants. The levels of production aresignificantly higher in cell supernatants for cells transfected with thesynthetic expression cassettes of the present invention.

Fractionation of the supernatants from mammalian cells transfected withthe synthetic Pol, Gag or Env expression cassette can be used to showthat the cassettes provide superior production of both Gag and Envproteins and, in the case of Gag, VLPs, relative to the wild-typesequences.

Efficient expression of these Pol, Gag- and/or Env-containingpolypeptides in mammalian cell lines provides the following benefits:the polypeptides are free of baculovirus contaminants; production byestablished methods approved by the FDA; increased purity; greateryields (relative to native coding sequences); and a novel method ofproducing the Pol, Gag- and/or Env-containing polypeptides in CHO cellswhich is not feasible in the absence of the increased expressionobtained using the constructs of the present invention. ExemplaryMammalian cell lines include, but are not limited to, BHK, VERO, HT1080,293, 293T, RD, COS-7, CHO, Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2,MT-4, H9, PM1, CEM, and CEMX174, such cell lines are available, forexample, from the A.T.C.C.).

A synthetic Gag expression cassette of the present invention will alsoexhibit high levels of expression and VLP production when transfectedinto insect cells. Synthetic Env expression cassettes also demonstratehigh levels of expression in insect cells. Further, in addition to ahigher total protein yield, the final product from the syntheticpolypeptides consistently contains lower amounts of contaminatingbaculovirus proteins than the final product from the native Pol, Gag orEnv.

Further, synthetic Pol, Gag and Env expression cassettes of the presentinvention can also be introduced into yeast vectors which, in turn, canbe transformed into and efficiently expressed by yeast cells(Saccharomyces cerevisea; using vectors as described in Rosenberg, S,and Tekamp-Olson, P., U.S. Pat. No. RE35,749, issued, Mar. 17, 1998,herein incorporated by reference).

In addition to the mammalian and insect vectors, the syntheticexpression cassettes of the present invention can be incorporated into avariety of expression vectors using selected expression controlelements. Appropriate vectors and control elements for any given celltype can be selected by one having ordinary skill in the art in view ofthe teachings of the present specification and information known in theart about expression vectors.

For example, a synthetic Pol, Gag or Env expression cassette can beinserted into a vector which includes control elements operably linkedto the desired coding sequence, which allow for the expression of thegene in a selected cell-type. For example, typical promoters formammalian cell expression include the SV40 early promoter, a CMVpromoter such as the CMV immediate early promoter (a CMV promoter caninclude intron A), RSV, HIV-Ltr, the mouse mammary tumor virus LTRpromoter (MMLV-ltr), the adenovirus major late promoter (Ad MLP), andthe herpes simplex virus promoter, among others. Other nonviralpromoters, such as a promoter derived from the murine metallothioneingene, will also find use for mammalian expression. Typically,transcription termination and polyadenylation sequences will also bepresent, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook, et al., supra, as well as a bovine growth hormoneterminator sequence. Introns, containing splice donor and acceptorsites, may also be designed into the constructs for use with the presentinvention (Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986),

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence (Chapman et al., Nuc. Acids Res.(1991) 19:3979-3986).

The desired synthetic Pol, Gag or Env polypeptide encoding sequences canbe cloned into any number of commercially available vectors to generateexpression of the polypeptide in an appropriate host system. Thesesystems include, but are not limited to, the following: baculovirusexpression {Reilly, P. R., et al., BACULOVIRUS EXPRESSION VECTORS: ALABORATORY MANUAL (1992); Beames, et al., Biotechniques 11:378 (1991);Pharmingen; Clontech, Palo Alto, Calif.)}, vaccinia expression {Earl, P.L., et al., “Expression of proteins in mammalian cells using vaccinia”In Current Protocols in Molecular Biology (F. M. Ausubel, et al. Eds.),Greene Publishing Associates & Wiley Interscience, New York (1991);Moss, B., et al., U.S. Pat. No. 5,135,855, issued 4 Aug. 1992},expression in bacteria {Ausubel, F. M., et al., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley and Sons, Inc., Media Pa.; Clontech},expression in yeast {Rosenberg, S, and Tekamp-Olson, P., U.S. Pat. No.RE35,749, issued, Mar. 17, 1998, herein incorporated by reference;Shuster, J. R., U.S. Pat. No. 5,629,203, issued May 13, 1997, hereinincorporated by reference; Gellissen, G., et al., Antonie VanLeeuwenhoek, 62(1-2):79-93 (1992); Romanos, M. A., et al., Yeast8(6):423-488 (1992); Goeddel, D. V., Methods in Enzymology 185 (1990);Guthrie, C., and G. R. Fink, Methods in Enzymology 194 (1991)1,expression in mammalian cells {Clontech; Gibco-BRL, Ground Island, N.Y.;e.g., Chinese hamster ovary (CHO) cell lines (Haynes, J., et al., Nuc.Acid. Res. 11:687-706 (1983); 1983, Lau, Y. F., et al., Mol. Cell. Biol.4:1469-1475 (1984); Kaufman, R. J., “Selection and coamplification ofheterologous genes in mammalian cells,” in Methods in Enzymology, vol.185, pp 537-566. Academic Press, Inc., San Diego Calif. (1991)1, andexpression in plant cells {plant cloning vectors, Clontech Laboratories,Inc., Palo Alto, Calif., and Pharmacia LKB Biotechnology, Inc.,Pistcataway, N J; Hood, E., et al., J. Bacteriol. 168:1291-1301 (1986);Nagel, R., et al., FEMS Microbiol. Lett. 67:325 (1990); An, et al.,“Binary Vectors”, and others in Plant Molecular Biology Manual A3:1-19(1988); Miki, B. L. A., et al., pp. 249-265, and others in Plant DNAInfectious Agents (Hohn, T., et al., eds.) Springer-Verlag, Wien,Austria, (1987); Plant Molecular Biology: Essential Techniques, P. G.Jones and J. M. Sutton, New York, J. Wiley, 1997; Miglani, GurbachanDictionary of Plant Genetics and Molecular Biology, New York, FoodProducts Press, 1998; Henry, R. J., Practical Applications of PlantMolecular Biology, New York, Chapman & Hall, 1997}.

Also included in the invention is an expression vector, containingcoding sequences and expression control elements which allow expressionof the coding regions in a suitable host. The control elements generallyinclude a promoter, translation initiation codon, and translation andtranscription termination sequences, and an insertion site forintroducing the insert into the vector. Translational control elementshave been reviewed by M. Kozak (e.g., Kozak, M., Mamm. Genome7(8):563-574, 1996; Kozak, M., Biochimie 76(9):815-821, 1994; Kozak, M.,J Cell Biol 108(2):229-241, 1989; Kozak, M., and Shatkin, A. J., MethodsEnzymol 60:360-375, 1979).

Expression in yeast systems has the advantage of commercial production.Recombinant protein production by vaccinia and CHO cell line have theadvantage of being mammalian expression systems. Further, vaccinia virusexpression has several advantages including the following: (i) its widehost range; (ii) faithful post-transcriptional modification, processing,folding, transport, secretion, and assembly of recombinant proteins;(iii) high level expression of relatively soluble recombinant proteins;and (iv) a large capacity to accommodate foreign DNA.

The recombinantly expressed polypeptides from synthetic Pol, Gag- and/orEnv-encoding expression cassettes are typically isolated from lysedcells or culture media. Purification can be carried out by methods knownin the art including salt fractionation, ion exchange chromatography,gel filtration, size-exclusion chromatography, size-fractionation, andaffinity chromatography. Immunoaffinity chromatography can be employedusing antibodies generated based on, for example, Gag or Env antigens.

Advantages of expressing the Pol, Gag- and/or Env-containing proteins ofthe present invention using mammalian cells include, but are not limitedto, the following: well-established protocols for scale-up production;the ability to produce VLPs; cell lines are suitable to meet goodmanufacturing process (GMP) standards; culture conditions for mammaliancells are known in the art.

Various forms of the different embodiments of the invention, describedherein, may be combined.

2.3 Production of Virus-Like Particles and Use of the Constructs of thePresent Invention to Create Packaging Cell Lines.

The group-specific antigens (Gag) of human immunodeficiency virus type-1(HIV-1) self-assemble into noninfectious virus-like particles (VLP) thatare released from various eucaryotic cells by budding (reviewed byFreed, E. O., Virology 251:1-15, 1998). The synthetic expressioncassettes of the present invention provide efficient means for theproduction of HIV-Gag virus-like particles (VLPs) using a variety ofdifferent cell types, including, but not limited to, mammalian cells.

Viral particles can be used as a matrix for the proper presentation ofan antigen entrapped or associated therewith to the immune system of thehost.

2.3.1 VLP Production Using the Synthetic Expression Cassettes of ThePresent Invention

Experiments can be performed in support of the present invention todemonstrate that the synthetic expression cassettes of the presentinvention provide superior production of both Gag proteins and VLPs,relative to native Gag coding sequences. Further, electron microscopicevaluation of VLP production can show that free and budding immaturevirus particles of the expected size are produced by cells containingthe synthetic expression cassettes.

Using the synthetic expression cassettes of the present invention,rather than native Gag coding sequences, for the production ofvirus-like particles provide several advantages. First, VLPs can beproduced in enhanced quantity making isolation and purification of theVLPs easier. Second, VLPs can be produced in a variety of cell typesusing the synthetic expression cassettes, in particular, mammalian celllines can be used for VLP production, for example, CHO cells. Productionusing CHO cells provides (i) VLP formation; (ii) correct myristylationand budding; (iii) absence of non-mammalian cell contaminants (e.g.,insect viruses and/or cells); and (iv) ease of purification. Thesynthetic expression cassettes of the present invention are also usefulfor enhanced expression in cell-types other than mammalian cell lines.For example, infection of insect cells with baculovirus vectors encodingthe synthetic expression cassettes results in higher levels of total Gagprotein yield and higher levels of VLP production (relative to wild-typecoding sequences). Further, the final product from insect cells infectedwith the baculovirus-Gag synthetic expression cassettes consistentlycontains lower amounts of contaminating insect proteins than the finalproduct when wild-type coding sequences are used.

VLPs can spontaneously form when the particle-forming polypeptide ofinterest is recombinantly expressed in an appropriate host cell. Thus,the VLPs produced using the synthetic expression cassettes of thepresent invention are conveniently prepared using recombinanttechniques. As discussed below, the Gag polypeptide encoding syntheticexpression cassettes of the present invention can include otherpolypeptide coding sequences of interest (for example, HIV protease, HIVpolymerase, HCV core; Env; synthetic Env; see, Example 1). Expression ofsuch synthetic expression cassettes yields VLPs comprising the Gagpolypeptide, as well as, the polypeptide of interest.

Once coding sequences for the desired particle-forming polypeptides havebeen isolated or synthesized, they can be cloned into any suitablevector or replicon for expression. Numerous cloning vectors are known tothose of skill in the art, and the selection of an appropriate cloningvector is a matter of choice. See, generally, Sambrook et al, supra. Thevector is then used to transform an appropriate host cell. Suitablerecombinant expression systems include, but are not limited to,bacterial, mammalian, baculovirus/insect, vaccinia, Semliki Forest virus(SFV), Alphaviruses (such as, Sindbis, Venezuelan Equine Encephalitis(VEE)), mammalian, yeast and Xenopus expression systems, well known inthe art. Particularly preferred expression systems are mammalian celllines, vaccinia, Sindbis, insect and yeast systems.

For example, a number of mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (A.T.C.C.), such as, but not limited to, Chinese hamsterovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkeykidney cells (COS), as well as others. Similarly, bacterial hosts suchas E. coli, Bacillus subtilis, and Streptococcus spp., will find usewith the present expression constructs. Yeast hosts useful in thepresent invention include inter alia, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, inter alia, Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni. See, e.g., Summers and Smith, TexasAgricultural Experiment Station Bulletin No. 1555 (1987).

Viral vectors can be used for the production of particles in eucaryoticcells, such as those derived from the pox family of viruses, includingvaccinia virus and avian poxvirus. Additionally, a vaccinia basedinfection/transfection system, as described in Tomei et al., J. Virol.(1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993)74:1103-1113, will also find use with the present invention. In thissystem, cells are first infected in vitro with a vaccinia virusrecombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the DNA of interest, driven by a T7 promoter. Thepolymerase expressed in the cytoplasm from the vaccinia virusrecombinant transcribes the transfected DNA into RNA which is thentranslated into protein by the host translational machinery.Alternately, T7 can be added as a purified protein or enzyme as in the“Progenitor” system (Studier and Moffatt, J. Mol. Biol. (1986)189:113-130). The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation product(s).

Depending on the expression system and host selected, the VLPS areproduced by growing host cells transformed by an expression vector underconditions whereby the particle-forming polypeptide is expressed andVLPs can be formed. The selection of the appropriate growth conditionsis within the skill of the art. If the VLPs are formed intracellularly,the cells are then disrupted, using chemical, physical or mechanicalmeans, which lyse the cells yet keep the VLPs substantially intact. Suchmethods are known to those of skill in the art and are described in,e.g., Protein Purification Applications: A Practical Approach, (E. L. V.Harris and S. Angal, Eds., 1990).

The particles are then isolated (or substantially purified) usingmethods that preserve the integrity thereof, such as, by gradientcentrifugation, e.g., cesium chloride (CsCl) sucrose gradients,pelleting and the like (see, e.g., Kirnbauer et al. J. Virol. (1993)67:6929-6936), as well as standard purification techniques including,e.g., ion exchange and gel filtration chromatography.

VLPs produced by cells containing the synthetic expression cassettes ofthe present invention can be used to elicit an immune response whenadministered to a subject. One advantage of the present invention isthat VLPs can be produced by mammalian cells carrying the syntheticexpression cassettes at levels previously not possible. As discussedabove, the VLPs can comprise a variety of antigens in addition to theGag polypeptide (e.g., Gag-protease, Gag-polymerase, Env, synthetic Env,etc.). Purified VLPs, produced using the synthetic expression cassettesof the present invention, can be administered to a vertebrate subject,usually in the form of vaccine compositions. Combination vaccines mayalso be used, where such vaccines contain, for example, an adjuvantsubunit protein (e.g., Env). Administration can take place using theVLPs formulated alone or formulated with other antigens; Further, theVLPs can be administered prior to, concurrent with, or subsequent to,delivery of the synthetic expression cassettes for DNA immunization (seebelow) and/or delivery of other vaccines. Also, the site of VLPadministration may be the same or different as other vaccinecompositions that are being administered. Gene delivery can beaccomplished by a number of methods including, but are not limited to,immunization with DNA, alphavirus vectors, pox virus vectors, andvaccinia virus vectors.

VLP immune-stimulating (or vaccine) compositions can include variousexcipients, adjuvants, carriers, auxiliary substances, modulatingagents, and the like. The immune stimulating compositions will includean amount of the VLP/antigen sufficient to mount an immunologicalresponse. An appropriate effective amount can be determined by one ofskill in the art. Such an amount will fall in a relatively broad rangethat can be determined through routine trials and will generally be anamount on the order of about 0.1 μg to about 1000 μg, more preferablyabout 1 μg to about 300 μg, of VLP/antigen.

A carrier is optionally present which is a molecule that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycollic acids, polymeric amino acids, amino acidcopolymers, lipid aggregates (such as oil droplets or liposomes), andinactive virus particles. Examples of particulate carriers include thosederived from polymethyl methacrylate polymers, as well as microparticlesderived from poly(lactides) and poly(lactide-co-glycolides), known asPLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee JP, et al., J Microencapsul. 14(2):197-210, 1997; O'Hagan D T, et al.,Vaccine 11(2):149-54, 1993. Such carriers are well known to those ofordinary skill in the art. Additionally, these carriers may function asimmunostimulating agents (“adjuvants”). Furthermore, the antigen may beconjugated to a bacterial toxoid, such as toxoid from diphtheria,tetanus, cholera, etc., as well as toxins derived from E. coli.

Adjuvants may also be used to enhance the effectiveness of thecompositions. Such adjuvants include, but are not limited to: (1)aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59 (International Publication No. WO 90/14837), containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing variousamounts of MTP-PE (see below), although not required) formulated intosubmicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP(see below) either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion, and (c) Ribi™ adjuvantsystem (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,0.2% Tween 80, and one or more bacterial cell wall components from thegroup consisting of monophosphorylipid A (MPL), trehalose dimycolate(TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3)saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester,Mass.) may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (6) oligonucleotides or polymeric moleculesencoding immunostimulatory CpG mofifs (Davis, H. L., et al., J.Immunology 160:870-876, 1998; Sato, Y. et al., Science 273:352-354,1996) or complexes of antigens/oligonucleotides {Polymeric moleculesinclude double and single stranded RNA and DNA, and backbonemodifications thereof, for example, methylphosphonate linkages; or (7)detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labiletoxin (LT), particularly LT-K63 (where lysine is substituted for thewild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S109 (whereserine is substituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (see, e.g.,International Publication Nos. WO93/13202 and W092/19265); and (8) othersubstances that act as immunostimulating agents to enhance theeffectiveness of the composition. Further, such polymeric moleculesinclude alternative polymer backbone structures such as, but not limitedto, polyvinyl backbones (Pitha, Biochem Biophys Acta, 204:39, 1970a;Pitha, Biopolymers, δ: 965, 1970b), and morpholino backbones (Summerton,J., et al., U.S. Pat. No. 5,142,047, issued Aug. 25, 1992; Summerton,J., et al., U.S. Pat. No. 5,185,444 issued Feb. 9, 1993). A variety ofother charged and uncharged polynucleotide analogs have been reported.Numerous backbone modifications are known in the art, including, but notlimited to, uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, and carbamates) and charged linkages(e.g., phosphorothioates and phosphorodithioates).}; and (7) othersubstances that act as immunostimulating agents to enhance theeffectiveness of the VLP immune-stimulating (or vaccine) composition.Alum, CpG oligonucleotides, and MF59 are preferred.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Dosage treatment with the VLP composition may be a single dose scheduleor a multiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals, chosen to maintainand/or reinforce the immune response, for example at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by theneed of the subject and be dependent on the judgment of thepractitioner.

If prevention of disease is desired, the antigen carrying VLPs aregenerally administered prior to primary infection with the pathogen ofinterest. If treatment is desired, e.g., the reduction of symptoms orrecurrences, the VLP compositions are generally administered subsequentto primary infection.

2.3.2 Using the Synthetic Expression Cassettes of the Present Inventionto Create Packaging Cell Lines

A number of viral based systems have been developed for use as genetransfer vectors for mammalian host cells. For example, retroviruses (inparticular, lentiviral vectors) provide a convenient platform for genedelivery systems. A coding sequence of interest (for example, a sequenceuseful for gene therapy applications) can be inserted into a genedelivery vector and packaged in retroviral particles using techniquesknown in the art. Recombinant virus can then be isolated and deliveredto cells of the subject either in vivo or ex vivo. A number ofretroviral systems have been described, including, for example, thefollowing: (U.S. Pat. No. 5,219,740; Miller et al. (1989) BioTechniques7:980; Miller, A. D. (1990) Human Gene Therapy 1:5; Scarpa et al. (1991)Virology 180:849; Burns et al. (1993) Proc. Natl. Acad. Sci. USA90:8033; Boris-Lawrie et al. (1993) Cur. Opin. Genet. Develop. 3:102; GB2200651; EP 0415731; EP 0345242; WO 89/02468; WO 89/05349; WO 89/09271;WO 90/02806; WO 90/07936; WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; WO 93/11230; WO 93/10218; WO 91/02805; in U.S. Pat. No.5,219,740; U.S. Pat. No. 4,405,712; U.S. Pat. No. 4,861,719; U.S. Pat.No. 4,980,289 and U.S. Pat. No. 4,777,127; in U.S. Ser. No. 07/800,921;and in Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res53:962-967; Ram (1993) Cancer Res 53:83-88; Takamiya (1992) Neurosci Res33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153;Cane (1984) Proc Natl Acad Sci USA 81; 6349; and Miller (1990) HumanGene Therapy 1.

In other embodiments, gene transfer vectors can be constructed to encodea cytokine or other immunomodulatory molecule. For example, nucleic acidsequences encoding native IL-2 and gamma-interferon can be obtained asdescribed in U.S. Pat. Nos. 4,738,927 and 5,326,859, respectively, whileuseful muteins of these proteins can be obtained as described in U.S.Pat. No. 4,853,332. Nucleic acid sequences encoding the short and longforms of mCSF can be obtained as described in U.S. Pat. Nos. 4,847,201and 4,879,227, respectively. In particular aspects of the invention,retroviral vectors expressing cytokine or immunomodulatory genes can beproduced as described herein (for example, employing the packaging celllines of the present invention) and in International Application No. PCTUS 94/02951, entitled “Compositions and Methods for CancerImmunotherapy.”

Examples of suitable immunomodulatory molecules for use herein includethe following: IL-1 and IL-2 (Karupiah et al. (1990) J. Immunology144:290-298, Weber et al. (1987) J. Exp. Med. 166:1716-1733, Gansbacheret al. (1990) J. Exp. Med. 172:1217-1224, and U.S. Pat. No. 4,738,927);IL-3 and IL-4 (Tepper et al. (1989) Cell 57:503-512, Golumbek et al.(1991) Science 254:713-716, and U.S. Pat. No. 5,017,691); IL-5 and IL-6(Brakenhof et al. (1987) J. Immunol. 139:4116-4121, and InternationalPublication No. WO 90/06370); IL-7 (U.S. Pat. No. 4,965,195); IL-8,IL-9, IL-10, IL-11, IL-12, and IL-13 (Cytokine Bulletin, Summer 1994);IL-14 and IL-15; alpha interferon (Finter et al. (1991) Drugs42:749-765, U.S. Pat. Nos. 4,892,743 and 4,966,843, InternationalPublication No. WO 85/02862, Nagata et al. (1980) Nature 284:316-320,Familletti et al. (1981) Methods in Enz. 78:387-394, Twu et al. (1989)Proc. Natl. Acad. Sci. USA 86:2046-2050, and Faktor et al. (1990)Oncogene 5:867-872); beta-interferon (Seif et al. (1991) J. Virol.65:664-671); gamma-interferons (Radford et al. (1991) The AmericanSociety of Hepatology 20082015, Watanabe et al. (1989) Proc. Natl. Acad.Sci. USA 86:9456-9460, Gansbacher et al. (1990) Cancer Research50:7820-7825, Maio et al. (1989) Can. Immunol. Immunother. 30:34-42, andU.S. Pat. Nos. 4,762,791 and 4,727,138); G-CSF (U.S. Pat. Nos. 4,999,291and 4,810,643); GM-CSF (International Publication No. WO 85/04188).

Immunomodulatory factors may also be agonists, antagonists, or ligandsfor these molecules. For example, soluble forms of receptors can oftenbehave as antagonists for these types of factors, as can mutated formsof the factors themselves.

Nucleic acid molecules that encode the above-described substances, aswell as other nucleic acid molecules that are advantageous for usewithin the present invention, may be readily obtained from a variety ofsources, including, for example, depositories such as the American TypeCulture Collection, or from commercial sources such as BritishBio-Technology Limited (Cowley, Oxford England). Representative examplesinclude BBG 12 (containing the GM-CSF gene coding for the mature proteinof 127 amino acids), BBG 6 (which contains sequences encoding gammainterferon), A.T.C.C. Deposit No. 39656 (which contains sequencesencoding TNF), A.T.C.C. Deposit No. 20663 (which contains sequencesencoding alpha-interferon), A.T.C.C. Deposit Nos. 31902, 31902 and 39517(which contain sequences encoding beta-interferon), A.T.C.C. Deposit No.67024 (which contains a sequence which encodes Interleukin-1b), A.T.C.C.Deposit Nos. 39405, 39452, 39516, 39626 and 39673 (which containsequences encoding Interleukin-2), A.T.C.C. Deposit Nos. 59399, 59398,and 67326 (which contain sequences encoding Interleukin-3), A.T.C.C.Deposit No. 57592 (which contains sequences encoding Interleukin-4),A.T.C.C. Deposit Nos. 59394 and 59395 (which contain sequences encodingInterleukin-5), and A.T.C.C. Deposit No. 67153 (which contains sequencesencoding Interleukin-6).

Plasmids containing cytokine genes or immunomodulatory genes(International Publication Nos. WO 94/02951 and WO 96/21015, both ofwhich are incorporated by reference in their entirety)can be digestedwith appropriate restriction enzymes, and DNA fragments containing theparticular gene of interest can be inserted into a gene transfer vectorusing standard molecular biology techniques. (See, e.g., Sambrook etal., supra., or Ausbel et al. (eds) Current Protocols in MolecularBiology, Greene Publishing and Wiley-Interscience).

Polynucleotide sequences coding for the above-described molecules can beobtained using recombinant methods, such as by screening cDNA andgenomic libraries from cells expressing the gene, or by deriving thegene from a vector known to include the same. For example, plasmidswhich contain sequences that encode altered cellular products may beobtained from a depository such as the A.T.C.C., or from commercialsources. Plasmids containing the nucleotide sequences of interest can bedigested with appropriate restriction enzymes, and DNA fragmentscontaining the nucleotide sequences can be inserted into a gene transfervector using standard molecular biology techniques.

Alternatively, cDNA sequences for use with the present invention may beobtained from cells which express or contain the sequences, usingstandard techniques, such as phenol extraction and PCR of cDNA orgenomic DNA. See, e.g., Sambrook et al., supra, for a description oftechniques used to obtain and isolate DNA. Briefly, mRNA from a cellwhich expresses the gene of interest can be reverse transcribed withreverse transcriptase using oligo-dT or random primers. The singlestranded cDNA may then be amplified by PCR (see U.S. Pat. Nos.4,683,202, 4,683,195 and 4,800,159, see also PCR Technology: Principlesand Applications for DNA Amplification, Erlich (ed.), Stockton Press,1989)) using oligonucleotide primers complementary to sequences oneither side of desired sequences.

The nucleotide sequence of interest can also be produced synthetically,rather than cloned, using a DNA synthesizer (e.g., an Applied BiosystemsModel 392 DNA Synthesizer, available from ABI, Foster City, Calif.). Thenucleotide sequence can be designed with the appropriate codons for theexpression product desired. The complete sequence is assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge (1981) Nature 292:756;Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311.

The synthetic expression cassettes of the present invention can beemployed in the construction of packaging cell lines for use withretroviral vectors.

One type of retrovirus, the murine leukemia virus, or “MLV”, has beenwidely utilized for gene therapy applications (see generally Maim et al.(Cell 33:153, 1993), Cane and Mulligan (Proc, Nat'l. Acad. Sci. USA81:6349, 1984), and Miller et al., Human Gene 2lerapy 1:5-14, 1990.

Lentiviral vectors typically, comprise a 5′ lentiviral LTR, a tRNAbinding site, a packaging signal, a promoter operably linked to one ormore genes of interest, an origin of second strand DNA synthesis and a3′ lentiviral LTR, wherein the lentiviral vector contains a nucleartransport element. The nuclear transport element may be located eitherupstream (5′) or downstream (3′) of a coding sequence of interest (forexample, a synthetic Gag or Env expression cassette of the presentinvention). Within certain embodiments, the nuclear transport element isnot RRE. Within one embodiment the packaging signal is an extendedpackaging signal. Within other embodiments the promoter is a tissuespecific promoter, or, alternatively, a promoter such as CMV. Withinother embodiments, the lentiviral vector further comprises an internalribosome entry site.

A wide variety of lentiviruses may be utilized within the context of thepresent invention, including for example, lentiviruses selected from thegroup consisting of HIV, HIV-1, HIV-2, FIV and SIV.

In one embodiment of the present invention synthetic Gag-polymeraseexpression cassettes are provided comprising a promoter and a sequenceencoding synthetic Gag-polymerase and at least one of vpr, vpu, nef orvif, wherein the promoter is operably linked to Gag-polymerase and vpr,vpu, nef or vif.

Within yet another aspect of the invention, host cells (e.g., packagingcell lines) are provided which contain any of the expression cassettesdescribed herein. For example, within one aspect packaging cell line areprovided comprising an expression cassette that comprises a sequenceencoding synthetic Gag-polymerase, and a nuclear transport element,wherein the promoter is operably linked to the sequence encodingGag-polymerase. Packaging cell lines may further comprise a promoter anda sequence encoding tat, rev, or an envelope, wherein the promoter isoperably linked to the sequence encoding tat, rev, Env or modified Envproteins. The packaging cell line may further comprise a sequenceencoding any one or more of nef, vif, vpu or vpr.

In one embodiment, the expression cassette (carrying, for example, thesynthetic Gag-polymerase) is stably integrated. The packaging cell line,upon introduction of a lentiviral vector, typically produces particles.The promoter regulating expression of the synthetic expression cassettemay be inducible. Typically, the packaging cell line, upon introductionof a lentiviral vector, produces particles that are essentially free ofreplication competent virus.

Packaging cell lines are provided comprising an expression cassettewhich directs the expression of a synthetic Gag-polymerase gene orcomprising an expression cassette which directs the expression of asynthetic Env genes described herein. (See, also, Andre, S., et al.,Journal of Virology 72(2):1497-1503, 1998; Haas, J., et al., CurrentBiology 6(3):315-324, 1996) for a description of other modified Envsequences). A lentiviral vector is introduced into the packaging cellline to produce a vector producing cell line.

As noted above, lentiviral vectors can be designed to carry or express aselected gene(s) or sequences of interest. Lentiviral vectors may bereadily constructed from a wide variety of lentiviruses (see RNA TumorViruses, Second Edition, Cold Spring Harbor Laboratory, 1985).Representative examples of lentiviruses included HIV, HIV-1, HIV-2, FIVand SIV. Such lentiviruses may either be obtained from patient isolates,or, more preferably, from depositories or collections such as theAmerican Type Culture Collection, or isolated from known sources usingavailable techniques.

Portions of the lentiviral gene delivery vectors (or vehicles) may bederived from different viruses. For example, in a given recombinantlentiviral vector, LTRs may be derived from an HIV, a packaging signalfrom SIV, and an origin of second strand synthesis from HrV-2.Lentiviral vector constructs may comprise a 5′ lentiviral LTR, a tRNAbinding site, a packaging signal, one or more heterologous sequences, anorigin of second strand DNA synthesis and a 3′ LTR, wherein saidlentiviral vector contains a nuclear transport element that is not RRE.

Briefly, Long Terminal Repeats (“LTRs”) are subdivided into threeelements, designated U5, R and U3. These elements contain a variety ofsignals which are responsible for the biological activity of aretrovirus, including for example, promoter and enhancer elements whichare located within U3. LTRs may be readily identified in the provirus(integrated DNA form) due to their precise duplication at either end ofthe genome. As utilized herein, a 5′ LTR should be understood to includea 5′ promoter element and sufficient LTR sequence to allow reversetranscription and integration of the DNA form of the vector. The 3′ LTRshould be understood to include a polyadenylation signal, and sufficientLTR sequence to allow reverse transcription and integration of the DNAform of the vector.

The tRNA binding site and origin of second strand DNA synthesis are alsoimportant for a retrovirus to be biologically active, and may be readilyidentified by one of skill in the art. For example, retroviral tRNAbinds to a tRNA binding site by Watson-Crick base pairing, and iscarried with the retrovirus genome into a viral particle. The tRNA isthen utilized as a primer for DNA synthesis by reverse transcriptase.The tRNA binding site may be readily identified based upon its locationjust downstream from the 5′LTR. Similarly, the origin of second strandDNA synthesis is, as its name implies, important for the second strandDNA synthesis of a retrovirus. This region, which is also referred to asthe poly-purine tract, is located just upstream of the 3′LTR.

In addition to a 5′ and 3′ LTR, tRNA binding site, and origin of secondstrand DNA synthesis, recombinant retroviral vector constructs may alsocomprise a packaging signal, as well as one or more genes or codingsequences of interest. In addition, the lentiviral vectors have anuclear transport element which, in preferred embodiments is not RRE.Representative examples of suitable nuclear transport elements includethe element in Rous sarcoma virus (Ogert, et al., J. ViroL 70,3834-3843, 1996), the element in Rous sarcoma virus (Liu & Mertz, Genes& Dev., 9, 1766-1789, 1995) and the element in the genome of simianretrovirus type I (Zolotukhin, et al., J. Virol. 68, 7944-7952, 1994).Other potential elements include the elements in the histone gene(Kedes, Annu. Rev. Biochem. 48, 837-870, 1970), the α-interferon gene(Nagata et al., Nature 287, 401-408, 1980), the β-adrenergic receptorgene (Koilka, et al., Nature 329, 75-79, 1987), and the c-Jun gene(Hattorie, et al., Proc. Natl. Acad. Sci. USA 85, 9148-9152, 1988).

Recombinant lentiviral vector constructs typically lack bothGag-polymerase and Env coding sequences. Recombinant lentiviral vectortypically contain less than 20, preferably 15, more preferably 10, andmost preferably 8 consecutive nucleotides found in Gag-polymerase andEnv genes. One advantage of the present invention is that the syntheticGag-polymerase expression cassettes, which can be used to constructpackaging cell lines for the recombinant retroviral vector constructs,have little homology to wild-type Gag-polymerase sequences and thusconsiderably reduce or eliminate the possibility of homologousrecombination between the synthetic and wild-type sequences.

Lentiviral vectors may also include tissue-specific promoters to driveexpression of one or more genes or sequences of interest.

Lentiviral vector constructs may be generated such that more than onegene of interest is expressed. This may be accomplished through the useof di- or oligo-cistronic cassettes (e.g., where the coding regions areseparated by 80 nucleotides or less, see generally Levin et al., Gene108:167-174, 1991), or through the use of Internal Ribosome Entry Sites(“IRES”).

Packaging cell lines suitable for use with the above describedrecombinant retroviral vector constructs may be readily prepared giventhe disclosure provided herein.

Briefly, the parent cell line from which the packaging cell line isderived can be selected from a variety of mammalian cell lines,including for example, 293, RD, COS-7, CHO, BHK, VERO HT1080, andmyeloma cells.

After selection of a suitable host cell for the generation of apackaging cell line, one or more expression cassettes are introducedinto the cell line in order to complement or supply in trans componentsof the vector which have been deleted.

Representative examples of suitable expression cassettes have beendescribed herein and include synthetic Env, synthetic Gag, syntheticGag-protease, and synthetic Gag-polymerase expression cassettes, whichcomprise a promoter and a sequence encoding, e.g., Gag-polymerase and atleast one of vpr, vpu, nef or vif, wherein the promoter is operablylinked to Gag-polymerase and vpr, vpu, nef or vif. As described above,the native and/or modified Env coding sequences may also be utilized inthese expression cassettes.

Utilizing the above-described expression cassettes, a wide variety ofpackaging cell lines can be generated. For example, within one aspectpackaging cell line are provided comprising an expression cassette thatcomprises a sequence encoding synthetic Gag-polymerase, and a nucleartransport element, wherein the promoter is operably linked to thesequence encoding Gag-polymerase. Within other aspects, packaging celllines are provided comprising a promoter and a sequence encoding tat,rev, Env, or other HIV antigens or epitopes derived therefrom, whereinthe promoter is operably linked to the sequence encoding tat, rev, Env,or the HIV antigen or epitope. Within further embodiments, the packagingcell line may comprise a sequence encoding any one or more of nef, vif,vpu or vpr. For example, the packaging cell line may contain only nef,vif, vpu, or vpr alone, nef and vif, nef and vpu, nef and vpr, vif andvpu, vif and vpr, vpu and vpr, nef vif and vpu, nef vif and vpr, nef vpuand vpr, vvir vpu and vpr, or, all four of nef vif vpu and vpr.

In one embodiment, the expression cassette is stably integrated. Withinanother embodiment, the packaging cell line, upon introduction of alentiviral vector, produces particles. Within further embodiments thepromoter is inducible. Within certain preferred embodiments of theinvention, the packaging cell line, upon introduction of a lentiviralvector, produces particles that are free of replication competent virus.

The synthetic cassettes containing optimized coding sequences aretransfected into a selected cell line. Transfected cells are selectedthat (i) carry, typically, integrated, stable copies of the Gag, Pol,and Env coding sequences, and (ii) are expressing acceptable levels ofthese polypeptides (expression can be evaluated by methods known in theprior art, e.g., see Examples 1-4). The ability of the cell line toproduce VLPs may also be verified.

A sequence of interest is constructed into a suitable viral vector asdiscussed above. This defective virus is then transfected into thepackaging cell line. The packaging cell line provides the viralfunctions necessary for producing virus-like particles into which thedefective viral genome, containing the sequence of interest, arepackaged. These VLPs are then isolated and can be used, for example, ingene delivery or gene therapy.

Further, such packaging cell lines can also be used to produce VLPsalone, which can, for example, be used as adjuvants for administrationwith other antigens or in vaccine compositions. Also, co-expression of aselected sequence of interest encoding a polypeptide (for example, anantigen) in the packaging cell line can also result in the entrapmentand/or association of the selected polypeptide in/with the VLPs.

Various forms of the different embodiments of the present invention(e.g., constructs) may be combined.

2.4 DNA Immunization and Gene Delivery

A variety of HIV polypeptide antigens, particularly Type C HIV antigens,can be used in the practice of the present invention. HIV antigens canbe included in DNA immunization constructs containing, for example, asynthetic Gag expression cassette fused in-frame to a coding sequencefor the polypeptide antigen, where expression of the construct resultsin VLPs presenting the antigen of interest.

HIV antigens of particular interest to be used in the practice of thepresent invention include tat, rev, nef, vif, vpu, vpr, and other HIVantigens or epitopes derived therefrom. For example, the packaging cellline may contain only nef, and HIV-1 (also known as HTLV-III, LAV, ARV,etc.), including, but not limited to, antigens such as gp120, gp41,gp160 (both native and modified); Gag; and pol from a variety ofisolates including, but not limited to, HIV_(IIIb), HIV_(SF2),HIV-1_(SF162), HIV-1_(SF170), HIV_(LAV), HIV_(LAI), HIV_(MN),HIV-1_(CM235), HIV-1_(US4), other HIV-1 strains from diverse subtypes(e.g., subtypes, A through G, and O), HIV-2 strains and diverse subtypes(e.g., HIV-2_(UC1) and HW-2_(UC2)). See, e.g., Myers, et al., Los AlamosDatabase, Los Alamos National Laboratory, Los Alamos, N. Mex.; Myers, etal., Human Retroviruses and Aids, 1990, Los Alamos, N. Mex. Los AlamosNational Laboratory.

To evaluate efficacy, DNA immunization using synthetic expressioncassettes of the present invention can be performed, for instance asdescribed in Example 4. Mice are immunized with both the Gag (and/orEnv) synthetic expression cassette and the Gag (and/or Env) wild typeexpression cassette. Mouse immunizations with plasmid-DNAs will showthat the synthetic expression cassettes provide a clear improvement ofimmunogenicity relative to the native expression cassettes. Also, thesecond boost immunization will induce a secondary immune response, forexample, after approximately two weeks. Further, the results of CTLassays will show increased potency of synthetic Gag (and/or Env)expression cassettes for induction of cytotoxic T-lymphocyte (CTL)responses by DNA immunization.

It is readily apparent that the subject invention can be used to mountan immune response to a wide variety of antigens and hence to treat orprevent a HIV infection, particularly Type C HIV infection.

2.4.1 Delivery of the Synthetic Expression Cassettes of the PresentInvention

Polynucleotide sequences coding for the above-described molecules can beobtained using recombinant methods, such as by screening cDNA andgenomic libraries from cells expressing the gene, or by deriving thegene from a vector known to include the same. Furthermore, the desiredgene can be isolated directly from cells and tissues containing thesame, using standard techniques, such as phenol extraction and PCR ofcDNA or genomic DNA. See, e.g., Sambrook et al., supra, for adescription of techniques used to obtain and isolate DNA. The gene ofinterest can also be produced synthetically, rather than cloned. Thenucleotide sequence can be designed with the appropriate codons for theparticular amino acid sequence desired. In general, one will selectpreferred codons for the intended host in which the sequence will beexpressed. The complete sequence is assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge, Nature (1981) 292:756;Nambair et al., Science (1984) 223:1299; Jay et al., J. Biol. Chem.(1984) 259:6311; Stemmer, W. P. C., (1995) Gene 164:49-53.

Next, the gene sequence encoding the desired antigen can be insertedinto a vector containing a synthetic Gag or synthetic Env expressioncassette of the present invention. The antigen is inserted into thesynthetic Gag coding sequence such that when the combined sequence isexpressed it results in the production of VLPs comprising the Gagpolypeptide and the antigen of interest, e.g., Env (native or modified)or other antigen derived from HIV. Insertions can be made within thecoding sequence or at either end of the coding sequence (5′, aminoterminus of the expressed Gag polypeptide; or 3′, carboxy terminus ofthe expressed Gag polypeptide)(Wagner, R., et al., Arch Virol.127:117-137, 1992; Wagner, R., et al., Virology 200:162-175, 1994; Wu,X., et al., J. Virol. 69(6):3389-3398, 1995; Wang, C-T., et al.,Virology 200:524-534, 1994; Chazal, N., et al., Virology 68(1):111-122,1994; Griffiths, J. C., et al., J. Virol. 67(6):3191-3198, 1993; Reicin,A. S., et al., J. Virol. 69(2):642-650, 1995).

Up to 50% of the coding sequences of p55Gag can be deleted withoutaffecting the assembly to virus-like particles and expression efficiency(Borsetti, A., et al, J. Virol. 72(11):9313-9317, 1998; Garnier, L., etal., J Virol 72(6):4667-4677, 1998; Zhang, Y., et al., J Virol72(3):1782-1789, 1998; Wang, C., et al., J Virol 72(10): 7950-7959,1998). In one embodiment of the present invention, immunogenicity of thehigh level expressing synthetic Gag expression cassettes can beincreased by the insertion of different structural or non-structural HIVantigens, multiepitope cassettes, or cytokine sequences into deletedregions of Gag sequence. Such deletions may be generated following theteachings of the present invention and information available to one ofordinary skill in the art. One possible advantage of this approach,relative to using full-length sequences fused to heterologouspolypeptides, can be higher expression/secretion efficiency of theexpression product.

When sequences are added to the amino terminal end of Gag, thepolynucletide can contain coding sequences at the 5′ end that encode asignal for addition of a myristic moiety to the Gag-containingpolypeptide (e.g., sequences that encode Met-Gly).

The ability of Gag-containing polypeptide constructs to form VLPs can beempirically determined following the teachings of the presentspecification.

Gag/antigen (e.g., Gag/Env) synthetic expression cassettes includecontrol elements operably linked to the coding sequence, which allow forthe expression of the gene in vivo in the subject species. For example,typical promoters for mammalian cell expression include the SV40 earlypromoter, a CMV promoter such as the CMV immediate early promoter, themouse mammary tumor virus LTR promoter, the adenovirus major latepromoter (Ad MLP), and the herpes simplex virus promoter, among others.Other nonviral promoters, such as a promoter derived from the murinemetallothionein gene, will also find use for mammalian expression.Typically, transcription termination and polyadenylation sequences willalso be present, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook et al., supra, as well as a bovine growth hormoneterminator sequence.

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence.

Furthermore, plasmids can be constructed which include a chimericantigen-coding gene sequences, encoding, e.g., multipleantigens/epitopes of interest, for example derived from more than oneviral isolate.

Typically the antigen coding sequences precede or follow the syntheticcoding sequence and the chimeric transcription unit will have a singleopen reading frame encoding both the antigen of interest and thesynthetic Gag coding sequences. Alternatively, multi-cistronic cassettes(e.g., bi-cistronic cassettes) can be constructed allowing expression ofmultiple antigens from a single mRNA using the EMCV IRES, or the like.

Once complete, the constructs are used for nucleic acid immunizationusing standard gene delivery protocols. Methods for gene delivery areknown in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859,5,589,466. Genes can be delivered either directly to the vertebratesubject or, alternatively, delivered ex vivo, to cells derived from thesubject and the cells reimplanted in the subject.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. Selected sequences can be insertedinto a vector and packaged in retroviral particles using techniquesknown in the art. The recombinant virus can then be isolated anddelivered to cells of the subject either in vivo or ex vivo. A number ofretroviral systems have been described (U.S. Pat. No. 5,219,740; Millerand Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human GeneTherapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burnset al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrieand Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.

A number of adenovirus vectors have also been described. Unlikeretroviruses which integrate into the host genome, adenoviruses persistextrachromosomally thus minimizing the risks associated with insertionalmutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett etal., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human GeneTherapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barret al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988)6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).

Additionally, various adeno-associated virus (AAV) vector systems havebeen developed for gene delivery. AAV vectors can be readily constructedusing techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070(published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993);Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al.,Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J.Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. CurrentTopics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. HumanGene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994)1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Another vector system useful for delivering the polynucleotides of thepresent invention is the enterically administered recombinant poxvirusvaccines described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

Additional viral vectors which will find use for delivering the nucleicacid molecules encoding the antigens of interest include those derivedfrom the pox family of viruses, including vaccinia virus and avianpoxvirus. By way of example, vaccinia virus recombinants expressing thegenes can be constructed as follows. The DNA encoding the particularsynthetic Gag/or Env/antigen coding sequence is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the codingsequences of interest into the viral genome. The resulting TKrecombinant can be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the genes. Recombinant avipox viruses,expressing immunogens from mammalian pathogens, are known to conferprotective immunity when administered to non-avian species. The use ofan avipox vector is particularly desirable in human and other mammalianspecies since members of the avipox genus can only productivelyreplicate in susceptible avian species and therefore are not infectivein mammalian cells. Methods for producing recombinant avipoxviruses areknown in the art and employ genetic recombination, as described abovewith respect to the production of vaccinia viruses. See, e.g., WO91/12882; WO 89/03429; and WO 92/03545.

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 andWagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can alsobe used for gene delivery.

Members of the Alphavirus genus, such as, but not limited to, vectorsderived from the Sindbis, Semliki Forest, and Venezuelan EquineEncephalitis viruses, will also find use as viral vectors for deliveringthe polynucleotides of the present invention (for example, a syntheticGag-polypeptide encoding expression cassette). For a description ofSindbis-virus derived vectors useful for the practice of the instantmethods, see, Dubensky et al., J. Virol. (1996) 70:508-519; andInternational Publication Nos. WO 95/07995 and WO 96/17072; as well as,Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec. 1,1998, and Dubensky, Jr., T. W., U.S. Pat. No. 5,789,245, issued Aug. 4,1998, both herein incorporated by reference.

A vaccinia based infection/transfection system can be conveniently usedto provide for inducible, transient expression of the coding sequencesof interest in a host cell. In this system, cells are first infected invitro with a vaccinia virus recombinant that encodes the bacteriophageT7 RNA polymerase. This polymerase displays exquisite specificity inthat it only transcribes templates bearing T7 promoters. Followinginfection, cells are transfected with the polynucleotide of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into protein by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

As an alternative approach to infection with vaccinia or avipox virusrecombinants, or to the delivery of genes using other viral vectors, anamplification system can be used that will lead to high level expressionfollowing introduction into host cells. Specifically, a T7 RNApolymerase promoter preceding the coding region for T7 RNA polymerasecan be engineered. Translation of RNA derived from this template willgenerate T7 RNA polymerase which in turn will transcribe more template.Concomitantly, there will be a cDNA whose expression is under thecontrol of the T7 promoter. Thus, some of the T7 RNA polymerasegenerated from translation of the amplification template RNA will leadto transcription of the desired gene. Because some T7 RNA polymerase isrequired to initiate the amplification, T7 RNA polymerase can beintroduced into cells along with the template(s) to prime thetranscription reaction. The polymerase can be introduced as a protein oron a plasmid encoding the RNA polymerase. For a further discussion of T7systems and their use for transforming cells, see, e.g., InternationalPublication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986)189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al.,Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc.Acids Res. (1993). 21:2867-2872; Chen et al., Nuc. Acids Res. (1994)22:2114-2120; and U.S. Pat. No. 5,135,855.

A synthetic Gag- and/or Env-containing expression cassette of interestcan also be delivered without a viral vector. For example, the syntheticexpression cassette can be packaged in liposomes prior to delivery tothe subject or to cells derived therefrom. Lipid encapsulation isgenerally accomplished using liposomes which are able to stably bind orentrap and retain nucleic acid. The ratio of condensed DNA to lipidpreparation can vary but will generally be around 1:1 (mg DNA:micromoleslipid), or more of lipid. For a review of the use of liposomes ascarriers for delivery of nucleic acids, see, Hug and Sleight, Biochim.Biophys. Acta. (1991) 1097:1-17; Straubinger et al., in Methods ofEnzymology (1983), Vol. 101, pp. 512-527.

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations, with cationic liposomes particularly preferred. Cationicliposomes have been shown to mediate intracellular delivery of plasmidDNA (Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416);mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081);and purified transcription factors (Debs et al., J. Biol. Chem. (1990)265:10189-10192), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987)84:7413-7416). Other commercially available lipids include (DDAB/DOPE)and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be preparedfrom readily available materials using techniques well known in the art.See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198;PCT Publication No. WO 90/11092 for a description of the synthesis ofDOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as,from Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY(1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA(1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta(1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA(1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szokaand Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; andSchaefer-Ridder et al., Science (1982) 215:166.

The DNA and/or protein antigen(s) can also be delivered in cochleatelipid compositions similar to those described by Papahadjopoulos et al.,Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos.4,663,161 and 4,871,488.

The synthetic expression cassette of interest may also be encapsulated,adsorbed to, or associated with, particulate carriers. Such carrierspresent multiple copies of a selected antigen to the immune system andpromote trapping and retention of antigens in local lymph nodes. Theparticles can be phagocytosed by macrophages and can enhance antigenpresentation through cytokine release. Examples of particulate carriersinclude those derived from polymethyl methacrylate polymers, as well asmicroparticles derived from poly(lactides) andpoly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al.,Pharm. Res. (1993) 10:362-368; McGee J P, et al., J Microencapsul.14(2):197-210, 1997; O'Hagan D T, et al., Vaccine 11(2):149-54, 1993.Suitable microparticles may also be manufactured in the presence ofcharged detergents, such as anionic or cationic detergents, to yieldmicroparticles with a surface having a net negative or a net positivecharge. For example, microparticles manufactured with anionicdetergents, such as hexadecyltrimethylammonium bromide (CTAB), i.e.CTAB-PLG microparticles, adsorb negatively charged macromolecules, suchas DNA. (see, e.g., Int'l Application Number PCT/US99/17308).

Furthermore, other particulate systems and polymers can be used for thein vivo or ex vivo delivery of the gene of interest. For example,polymers such as polylysine, polyarginine, polyornithine, spermine,spermidine, as well as conjugates of these molecules, are useful fortransferring a nucleic acid of interest. Similarly, DEAEdextran-mediated transfection, calcium phosphate precipitation orprecipitation using other insoluble inorganic salts, such as strontiumphosphate, aluminum silicates including bentonite and kaolin, chromicoxide, magnesium silicate, talc, and the like, will find use with thepresent methods. See, e.g., Feigner, P. L., Advanced Drug DeliveryReviews (1990) 5:163-187, for a review of delivery systems useful forgene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No.5,831,005, issued Nov. 3, 1998, herein incorporated by reference) mayalso be used for delivery of a construct of the present invention.

Additionally, biolistic delivery systems employing particulate carrierssuch as gold and tungsten, are especially useful for deliveringsynthetic expression cassettes of the present invention. The particlesare coated with the synthetic expression cassette(s) to be delivered andaccelerated to high velocity, generally under a reduced atmosphere,using a gun powder discharge from a “gene gun.” For a description ofsuch techniques, and apparatuses useful therefore, see, e.g., U.S. Pat.Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and5,478,744. Also, needle-less injection systems can be used (Davis, H.L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

Recombinant vectors carrying a synthetic expression cassette of thepresent invention are formulated into compositions for delivery to thevertebrate subject. These compositions may either be prophylactic (toprevent infection) or therapeutic (to treat disease after infection).The compositions will comprise a “therapeutically effective amount” ofthe gene of interest such that an amount of the antigen can be producedin vivo so that an immune response is generated in the individual towhich it is administered. The exact amount necessary will vary dependingon the subject being treated; the age and general condition of thesubject to be treated; the capacity of the subject's immune system tosynthesize antibodies; the degree of protection desired; the severity ofthe condition being treated; the particular antigen selected and itsmode of administration, among other factors. An appropriate effectiveamount can be readily determined by one of skill in the art. Thus, a“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through routine trials.

The compositions will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present in such vehicles.Certain facilitators of nucleic acid uptake and/or expression can alsobe included in the compositions or coadministered, such as, but notlimited to, bupivacaine, cardiotoxin and sucrose.

Once formulated, the compositions of the invention can be administereddirectly to the subject (e.g., as described above) or, alternatively,delivered ex vivo, to cells derived from the subject, using methods suchas those described above. For example, methods for the ex vivo deliveryand reimplantation of transformed cells into a subject are known in theart and can include, e.g., dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, lipofectamineand LT-1 mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) (with or without thecorresponding antigen) in liposomes, and direct microinjection of theDNA into nuclei.

Direct delivery of synthetic expression cassette compositions in vivowill generally be accomplished with or without viral vectors, asdescribed above, by injection using either a conventional syringe or agene gun, such as the Accell® gene delivery system (PowderJectTechnologies, Inc., Oxford, England). The constructs can be injectedeither subcutaneously, epidermally, intradermally, intramucosally suchas nasally, rectally and vaginally, intraperitoneally, intravenously,orally or intramuscularly. Delivery of DNA into cells of the epidermisis particularly preferred as this mode of administration provides accessto skin-associated lymphoid cells and provides for a transient presenceof DNA in the recipient. Other modes of administration include oral andpulmonary administration, suppositories, needle-less injection,transcutaneous and transdermal applications. Dosage treatment may be asingle dose schedule or a multiple dose schedule. Administration ofnucleic acids may also be combined with administration of peptides orother substances.

2.4.2 Ex Vivo Delivery of the Synthetic Expression Cassettes of thePresent Invention

In one embodiment, T cells, and related cell types (including but notlimited to antigen presenting cells, such as, macrophage, monocytes,lymphoid cells, dendritic cells, B-cells, T-cells, stem cells, andprogenitor cells thereof), can be used for ex vivo delivery of thesynthetic expression cassettes of the present invention. T cells can beisolated from peripheral blood lymphocytes (PBLs) by a variety ofprocedures known to those skilled in the art. For example, T cellpopulations can be “enriched” from a population of PBLs through theremoval of accessory and B cells. In particular, T cell enrichment canbe accomplished by the elimination of non-T cells using anti-MHC classII monoclonal antibodies. Similarly, other antibodies can be used todeplete specific populations of non-T cells. For example, anti-Igantibody molecules can be used to deplete B cells and anti-MacI antibodymolecules can be used to deplete macrophages.

T cells can be further fractionated into a number of differentsubpopulations by techniques known to those skilled in the art. Twomajor subpopulations can be isolated based on their differentialexpression of the cell surface markers CD4 and CD8. For example,following the enrichment of T cells as described above, CD4⁺ cells canbe enriched using antibodies specific for CD4 (see Coligan et al.,supra). The antibodies may be coupled to a solid support such asmagnetic beads. Conversely, CD8+ cells can be enriched through the useof antibodies specific for CD4 (to remove CD4⁺ cells), or can beisolated by the use of CD8 antibodies coupled to a solid support. CD4lymphocytes from HIV-1 infected patients can be expanded ex vivo, beforeor after transduction as described by Wilson et. al. (1995) J. Infect.Dis. 172:88.

Following purification of T cells, a variety of methods of geneticmodification known to those skilled in the art can be performed usingnon-viral or viral-based gene transfer vectors constructed as describedherein. For example, one such approach involves transduction of thepurified T cell population with vector-containing supernatant ofcultures derived from vector producing cells. A second approach involvesco-cultivation of an irradiated monolayer of vector-producing cells withthe purified T cells. A third approach involves a similar co-cultivationapproach; however, the purified T cells are pre-stimulated with variouscytokines and cultured 48 hours prior to the co-cultivation with theirradiated vector producing cells. Pre-stimulation prior to suchtransduction increases effective gene transfer (Nolta et al. (1992) Exp.Hematol. 20:1065). Stimulation of these cultures to proliferate alsoprovides increased cell populations for re-infusion into the patient.Subsequent to co-cultivation, T cells are collected from the vectorproducing cell monolayer, expanded, and frozen in liquid nitrogen.

Gene transfer vectors, containing one or more synthetic expressioncassette of the present invention (associated with appropriate controlelements for delivery to the isolated T cells) can be assembled usingknown methods.

Selectable markers can also be used in the construction of gene transfervectors. For example, a marker can be used which imparts to a mammaliancell transduced with the gene transfer vector resistance to a cytotoxicagent. The cytotoxic agent can be, but is not limited to, neomycin,aminoglycoside, tetracycline, chloramphenicol, sulfonamide, actinomycin,netropsin, distamycin A, anthracycline, or pyrazinamide. For example,neomycin phosphotransferase II imparts resistance to the neomycinanalogue geneticin (G418).

The T cells can also be maintained in a medium containing at least onetype of growth factor prior to being selected. A variety of growthfactors are known in the art which sustain the growth of a particularcell type. Examples of such growth factors are cytokine mitogens such asrIL-2, IL-10, IL-12, and IL-15, which promote growth and activation oflymphocytes. Certain types of cells are stimulated by other growthfactors such as hormones, including human chorionic gonadotropin (hCG)and human growth hormone. The selection of an appropriate growth factorfor a particular cell population is readily accomplished by one of skillin the art.

For example, white blood cells such as differentiated progenitor andstem cells are stimulated by a variety of growth factors. Moreparticularly, IL-3, IL-4, IL-5, IL-6, IL-9, GM-CSF, M-CSF, and G-CSF,produced by activated T_(H) and activated macrophages, stimulate myeloidstem cells, which then differentiate into pluripotent stem cells,granulocyte-monocyte progenitors, eosinophil progenitors, basophilprogenitors, megakaryocytes, and erythroid progenitors. Differentiationis modulated by growth factors such as GM-CSF, IL-3, IL-6, IL-11, andEPO.

Pluripotent stem cells then differentiate into lymphoid stem cells, bonemarrow stromal cells, T cell progenitors, B cell progenitors,thymocytes, T_(H) Cells, T_(C) cells, and B cells. This differentiationis modulated by growth factors such as IL-3, IL-4, IL-6, IL-7, GM-CSF,M-CSF, G-CSF, IL-2, and IL-5.

Granulocyte-monocyte progenitors differentiate to monocytes,macrophages, and neutrophils. Such differentiation is modulated by thegrowth factors GM-CSF, M-CSF, and IL-8. Eosinophil progenitorsdifferentiate into eosinophils. This process is modulated by GM-CSF andIL-5.

The differentiation of basophil progenitors into mast cells andbasophils is modulated by GM-CSF, IL-4, and IL-9. Megakaryocytes produceplatelets in response to GM-CSF, EPO, and IL-6. Erythroid progenitorcells differentiate into red blood cells in response to EPO.

Thus, during activation by the CD3-binding agent, T cells can also becontacted with a mitogen, for example a cytokine such as IL-2. Inparticularly preferred embodiments, the IL-2 is added to the populationof T cells at a concentration of about 50 to 100 μg/ml. Activation withthe CD3-binding agent can be carried out for 2 to 4 days.

Once suitably activated, the T cells are genetically modified bycontacting the same with a suitable gene transfer vector underconditions that allow for transfection of the vectors into the T cells.Genetic modification is carried out when the cell density of the T cellpopulation is between about 0.1×10⁶ and 5×10⁶, preferably between about0.5×10⁶ and 2×10⁶. A number of suitable viral and nonviral-based genetransfer vectors have been described for use herein.

After transduction, transduced cells are selected away fromnon-transduced cells using known techniques. For example, if the genetransfer vector used in the transduction includes a selectable markerwhich confers resistance to a cytotoxic agent, the cells can becontacted with the appropriate cytotoxic agent, whereby non-transducedcells can be negatively selected away from the transduced cells. If theselectable marker is a cell surface marker, the cells can be contactedwith a binding agent specific for the particular cell surface marker,whereby the transduced cells can be positively selected away from thepopulation. The selection step can also entail fluorescence-activatedcell sorting (FACS) techniques, such as where FACS is used to selectcells from the population containing a particular surface marker, or theselection step can entail the use of magnetically responsive particlesas retrievable supports for target cell capture and/or backgroundremoval.

More particularly, positive selection of the transduced cells can beperformed using a FACS cell sorter (e.g. a FACSVantage™ Cell Sorter,Becton Dickinson Immunocytometry Systems, San Jose, Calif.) to sort andcollect transduced cells expressing a selectable cell surface marker.Following transduction, the cells are stained with fluorescent-labeledantibody molecules directed against the particular cell surface marker.The amount of bound antibody on each cell can be measured by passingdroplets containing the cells through the cell sorter. By imparting anelectromagnetic charge to droplets containing the stained cells, thetransduced cells can be separated from other cells. The positivelyselected cells are then harvested in sterile collection vessels. Thesecell sorting procedures are described in detail, for example, in theFACSVantage™ Training Manual, with particular reference to sections 3-11to 3-28 and 10-1 to 10-17.

Positive selection of the transduced cells can also be performed usingmagnetic separation of cells based on expression or a particular cellsurface marker. In such separation techniques, cells to be positivelyselected are first contacted with specific binding agent (e.g., anantibody or reagent the interacts specifically with the cell surfacemarker). The cells are then contacted with retrievable particles (e.g.,magnetically responsive particles) which are coupled with a reagent thatbinds the specific binding agent (that has bound to the positive cells).The cell-binding agent-particle complex can then be physically separatedfrom non-labeled cells, for example using a magnetic field. When usingmagnetically responsive particles, the labeled cells can be retained ina container using a magnetic filed while the negative cells are removed.These and similar separation procedures are known to those of ordinaryskill in the art.

Expression of the vector in the selected transduced cells can beassessed by a number of assays known to those skilled in the art. Forexample, Western blot or Northern analysis can be employed depending onthe nature of the inserted nucleotide sequence of interest. Onceexpression has been established and the transformed T cells have beentested for the presence of the selected synthetic expression cassette,they are ready for infusion into a patient via the peripheral bloodstream.

The invention includes a kit for genetic modification of an ex vivopopulation of primary mammalian cells. The kit typically contains a genetransfer vector coding for at least one selectable marker and at leastone synthetic expression cassette contained in one or more containers,ancillary reagents or hardware, and instructions for use of the kit.

EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Generation of Synthetic Expression Cassettes

A. Modification of HIV-1 Env, Gag, Pol Nucleic Acid Coding Sequences

The Pol coding sequences were selected from Type C strain AF110975. TheGag coding sequences were selected from the Type C strains AF110965 andAF110967. The Env coding sequences were selected from Type C strainsAF110968 and AF110975. These sequences were manipulated to maximizeexpression of their gene products.

First, the HIV-1 codon usage pattern was modified so that the resultingnucleic acid coding sequence was comparable to codon usage found inhighly expressed human genes. The HIV codon usage reflects a highcontent of the nucleotides A or T of the codon-triplet. The effect ofthe HIV-1 codon usage is a high AT content in the DNA sequence thatresults in a decreased translation ability and instability of the mRNA.In comparison, highly expressed human codons prefer the nucleotides G orC. The coding sequences were modified to be comparable to codon usagefound in highly expressed human genes.

Second, there are inhibitory (or instability) elements (INS) locatedwithin the coding sequences of the Gag and Gag-protease coding sequences(Schneider R, et al., J. Virol. 71(7):4892-4903, 1997). RRE is asecondary RNA structure that interacts with the HIV encoded Rev-proteinto overcome the expression down-regulating effects of the INS. Toovercome the post-transcriptional activating mechanisms of RRE and Rev,the instability elements are inactivated by introducing multiple pointmutations that do not alter the reading frame of the encoded proteins.FIGS. 5 and 6 (SEQ ID Nos: 3, 4, 20 and 21) show the location of someremaining INS in synthetic sequences derived from strains AF110965 andAF110967. The changes made to these sequences are boxed in the Figures.In FIGS. 5 and 6, the top line depicts a codon optimized sequence of Gagpolypeptides from the indicated strains. The nucleotide(s) appearingbelow the line in the boxed region(s) depicts changes made to furtherremove INS. Thus, when the changes indicated in the boxed regions aremade, the resulting sequences correspond to the sequences depicted inFIGS. 1 and 2, respectively.

The synthetic coding sequences are assembled by methods known in theart, for example by companies such as the Midland Certified ReagentCompany (Midland, Tex.).

In one embodiment of the invention, sequences encoding Pol-polypeptidesare included with the synthetic Gag or Env sequences in order toincrease the number of epitopes for virus-like particles expressed bythe synthetic, optimized Gag/Env expression cassette. Because syntheticHIV-1 Pol expresses the functional enzymes reverse transcriptase (RT)and integrase (INT) (in addition to the structural proteins andprotease), it may be helpful in some instances to inactivate RT and INTfunctions. Several deletions or mutations in the RT and INT codingregions can be made to achieve catalytic nonfunctional enzymes withrespect to their RT and INT activity. {Jay. A. Levy (Editor) (1995) TheRetroviridae, Plenum Press, New York. ISBN 0-306-45033X. Pages 215-20;Grimison, B. and Laurence, J. (1995), Journal Of Acquired ImmuneDeficiency Syndromes and Human Retrovirology 9(1):58-68; Wakefield, J.K., et al., (1992) Journal Of Virology 66(11):6806-6812; Esnouf, R., etal., (1995) Nature Structural Biology 2(4):303-308; Maignan, S., et al.,(1998) Journal Of Molecular Biology 282(2):359-368; Katz, R. A. andSkalka, A. M. (1994) Annual Review Of Biochemistry 73 (1994);Jacobo-Molina, A., et al., (1993) Proceedings Of the National Academy OfSciences Of the United States Of America 90(13):6320-6324; Hickman, A.B., et al., (1994) Journal Of Biological Chemistry 269(46):29279-29287;Goldgur, Y., et al., (1998) Proceedings Of the National Academy OfSciences Of the United States Of America 95(16):9150-9154; Goette, M.,et al., (1998) Journal Of Biological Chemistry 273(17):10139-10146;Gorton, J. L., et al., (1998) Journal of Virology 72(6):5046-5055;Engelman, A., et al., (1997) Journal Of Virology 71(5):3507-3514; Dyda,F., et al., Science 266(5193):1981-1986; Davies, J. F., et al., (1991)Science 252(5002):88-95; Bujacz, G., et al., (1996) Febs Letters398(2-3):175-178; Beard, W. A., et al., (1996) Journal Of BiologicalChemistry 271(21):12213-12220; Kohlstaedt, L. A., et al., (1992) Science256(5065):1783-1790; Krug, M. S, and Berger, S. L. (1991) Biochemistry30(44):10614-10623; Mazumder, A., et al., (1996) Molecular Pharmacology49(4):621-628; Palaniappan, C., et al., (1997) Journal Of BiologicalChemistry 272(17):11157-11164; Rodgers, D. W., et al., (1995)Proceedings Of the National Academy Of Sciences Of the United States OfAmerica 92(4):1222-1226; Sheng, N. and Dennis, D. (1993) Biochemistry32(18):4938-4942; Spence, R. A., et al., (1995) Science267(5200):988-993}.

Furthermore selected B- and/or T-cell epitopes can be added to the Polconstructs (e.g., 3′ of the truncated INT or within the deletions of theRT- and INT-coding sequence) to replace and augment any epitopes deletedby the functional modifications of RT and INT. Alternately, selected B-and T-cell epitopes (including CTL epitopes) from RT and INT can beincluded in a minimal VLP formed by expression of the synthetic Gag orsynthetic Pol cassette, described above. (For descriptions of known HIVB- and T-cell epitopes see, HIV Molecular Immunology Database CTL SearchInterface; Los Alamos Sequence Compendia, 1987-1997; Internet address:http://hiv-web.lanl.gov/immunology/index.html.)

The resulting modified coding sequences are presented as a synthetic Envexpression cassette; a synthetic Gag expression cassette; a syntheticPol expression cassette. A common Gag region (Gag-common) extends fromnucleotide position 844 to position 903 (SEQ ID NO:1), relative toAF110965 (or from approximately amino acid residues 282 to 301 of SEQ IDNO:17) and from nucleotide position 841 to position 900 (SEQ ID NO:2),relative to AF110967 (or from approximately amino acid residues 281 to300 of SEQ ID NO:22). A common Env region (Env-common) extends fromnucleotide position 1213 to position 1353 (SEQ ID NO:5) and amino acidpositions 405 to 451 of SEQ ID NO:23, relative to AF110968 and fromnucleotide position 1210 to position 1353 (SEQ ID NO:11) and amino acidpositions 404-451 (SEQ ID NO:24), relative to

AF110975.

The synthetic DNA fragments for Pol, Gag and Env are cloned into thefollowing eucaryotic expression vectors: pCMVKm2, for transientexpression assays and DNA immunization studies, the pCMVKm2 vector isderived from pCMV6a (Chapman et al., Nuc. Acids Res. (1991)19:3979-3986) and comprises a kanamycin selectable marker, a ColE1origin of replication, a CMV promoter enhancer and Intron A, followed byan insertion site for the synthetic sequences described below followedby a polyadenylation signal derived from bovine growth hormone—thepCMVKm2 vector differs from the pCMV-link vector only in that apolylinker site is inserted into pCMVKm2 to generate pCMV-link;pESN2dhfr and pCMVPLEdhfr, for expression in Chinese Hamster Ovary (CHO)cells; and, pAcC13, a shuttle vector for use in the Baculovirusexpression system (pAcC13, is derived from pAcC12 which is described byMunemitsu S., et al., Mol Cell Biol. 10(145977-5982, 1990).

Briefly, construction of pCMVPLEdhfr was as follows.

To construct a DHFR cassette, the EMCV IRES (internal ribosome entrysite) leader was PCR-amplified from pCite-4a+ (Novagen, Inc., Milwaukee,Wis.) and inserted into pET-23d (Novagen, Inc., Milwaukee, Wis.) as anXba-Nco fragment to give pET-EMCV. The dhfr gene was PCR-amplified frompESN2dhfr to give a product with a Gly-Gly-Gly-Ser (SEQ ID NO: 46)spacer in place of the translation stop codon and inserted as anNco-BamH1 fragment to give pET-E-DHFR. Next, the attenuated neo gene wasPCR amplified from a pSV2Neo (Clontech, Palo Alto, Calif.) derivativeand inserted into the unique BamH1 site of pET-E-DHFR to givepET-E-DHFR/Neo(m2). Finally the bovine growth hormone terminator frompcDNA3 (Invitrogen, Inc., Carlsbad, Calif.) was inserted downstream ofthe neo gene to give pET-E-DHFR/Neo(m2)BGHt. The EMCV-dhfr/neoselectable marker cassette fragment was prepared by cleavage ofpET-E-DHFR/Neo(m2)BGHt.

The CMV enhancer/promoter plus Intron A was transferred from pCMV6a(Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986) as a HindIII-SalIfragment into pUC19 (New England Biolabs, Inc., Beverly, Mass.). Thevector backbone of pUC19 was deleted from the NdeI to the Sapl sites.The above described DHFR cassette was added to the construct such thatthe EMCV IRES followed the CMV promoter. The vector also contained anampr gene and an SV40 origin of replication.

B. Defining of the Major Homology Region (MHR) of HIV-1 p55Gag

The Major Homology Region (MHR) of HIV-1 p55 (Gag) is located in thep24-CA sequence of Gag. It is a conserved stretch of approximately 20amino acids. The position in the wild type AF110965 Gag protein is from282-301 (SEQ ID NO:25) and spans a region from 844-903 (SEQ ID NO:26)for the Gag DNA-sequence. The position in the synthetic Gag protein isalso from 282-301 (SEQ ID NO:25) and spans a region from 844-903 (SEQ IDNO:1) for the synthetic Gag DNA-sequence. The position in the wild typeand synthetic AF110967 Gag protein is from 281-300 (SEQ ID NO:27) andspans a region from 841-900 (SEQ ID NO:2) for the modified GagDNA-sequence. Mutations or deletions in the MHR can severely impairparticle production (Borsetti, A., et al., J. Virol. 72(11):9313-9317,1998; Mammano, F., et al., J Virol 68(8):4927-4936, 1994).

Percent identity to this sequence can be determined, for example, usingthe Smith-Waterman search algorithm (Time Logic, Incline Village, NV),with the following exemplary parameters: weight matrix=nuc4×4hb; gapopening penalty=20, gap extension penalty=5.

C. Defining of the Common Sequence Region of HIV-1 Env

The common sequence region (CSR) of HIV-1 Env is located in the C4sequence of Env. It is a conserved stretch of approximately 47 aminoacids. The position in the wild type and synthetic AF110968 Env proteinis from approximately amino acid residue 405 to 451 (SEQ ID NO:28) andspans a region from 1213 to 1353 (SEQ ID NO:5) for the Env DNA-sequence.The position in the wild type and synthetic AF110975 Env protein is fromapproximately amino acid residue 404 to 451 (SEQ ID NO:29) and spans aregion from 1210 to 1353 (SEQ ID NO:11) for the Env DNA-sequence.

Percent identity to this sequence can be determined, for example, usingthe Smith-Waterman search algorithm (Time Logic, Incline Village, NV),with the following exemplary parameters: weight matrix=nuc4×4hb; gapopening penalty=20, gap extension penalty=5.

Various forms of the different embodiments of the invention, describedherein, may be combined.

Example 2 Expression Assays for the Synthetic Coding Sequences

A. Env, Gag and Gag-Protease Coding Sequences

The wild-type Pol (from AF110975), Env (from AF110968 or AF110975) andGag (from AF110965 and AF110967) sequences are cloned into expressionvectors having the same features as the vectors into which the syntheticPol, Env and Gag and sequences are cloned.

Expression efficiencies for various vectors carrying the wild-type andsynthetic Pol, Env and Gag sequences are evaluated as follows. Cellsfrom several mammalian cell lines (293, RD, COS-7, and CHO; all obtainedfrom the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209) are transfected with 2 μg of DNA intransfection reagent LT1 (PanVera Corporation, 545 Science Dr., Madison,Wis.). The cells are incubated for 5 hours in reduced serum medium(Opti-MEM, Gibco-BRL, Gaithersburg, Md.). The medium is then replacedwith normal medium as follows: 293 cells, IMDM, 10% fetal'calf serum, 2%glutamine (BioWhittaker, Walkersville, Md.); RD and COS-7 cells, D-MEM,10% fetal calf serum, 2% glutamine (Opti-MEM, Gibco-BRL, Gaithersburg,Md.); and CHO cells, Ham's F-12, 10% fetal calf serum, 2% glutamine(Opti-MEM, Gibco-BRL, Gaithersburg, Md.). The cells are incubated foreither 48 or 60 hours. Cell lysates are collected as described below inExample 3. Supernatants are harvested and filtered through 0.45 pmsyringe filters. Supernatants are evaluated using the Coulter p24-assay(Coulter Corporation, Hialeah, Fla., US), using 96-well plates coatedwith a murine monoclonal antibody directed against HIV core antigen. TheHIV-1 p24 antigen binds to the coated wells. Biotinylated antibodiesagainst HIV recognize the bound p24 antigen. Conjugatedstrepavidin-horseradish peroxidase reacts with the biotin. Colordevelops from the reaction of peroxidase with TMB substrate. Thereaction is terminated by addition of 4N H₂SO₄. The intensity of thecolor is directly proportional to the amount of HIV p24 antigen in asample.

Synthetic Pol, Env, Gag expression cassettes provides dramatic increasesin production of their protein products, relative to the native(wild-type Type C) sequences, when expressed in a variety of cell lines.

Example 3 Western Blot Analysis of Expression

A. Env, Gag and Pol Coding Sequences

Human 293 cells are transfected as described in Example 2 withpCMV6a-based vectors containing native or synthetic Pol, Env or Gagexpression cassettes. Cells are cultivated for 60 hourspost-transfection. Supernatants are prepared as described. Cell lysatesare prepared as follows. The cells are washed once withphosphate-buffered saline, lysed with detergent [1% NP40 (Sigma ChemicalCo., St. Louis, Mo.) in 0.1 M Tris-HCl, pH 7.5], and the lysatetransferred into fresh tubes. SDS-polyacrylamide gels (pre-cast 8-16%;Novex, San Diego, Calif.) are loaded with 20 μl of supernatant or 12.5μl of cell lysate. A protein standard is also loaded (5 μl, broad sizerange standard; BioRad Laboratories, Hercules, Calif.). Electrophoresisis carried out and the proteins are transferred using a BioRad TransferChamber (BioRad Laboratories, Hercules, Calif.) to Immobilon P membranes(Millipore Corp., Bedford, Mass.) using the transfer buffer recommendedby the manufacturer (Millipore), where the transfer is performed at 100volts for 90 minutes. The membranes are exposed to HIV-1-positive humanpatient serum and immunostained using o-phenylenediamine dihydrochloride(OPD; Sigma).

Immunoblotting analysis shows that cells containing the synthetic Pol,Env or Gag expression cassette produce the expected protein at higherper-cell concentrations than cells containing the native expressioncassette. The proteins are seen in both cell lysates and supernatants.The levels of production are significantly higher in cell supernatantsfor cells transfected with the synthetic expression cassettes of thepresent invention.

In addition, supernatants from the transfected 293 cells arefractionated on sucrose gradients. Aliquots of the supernatant aretransferred to Polyclear™ ultra-centrifuge tubes (Beckman Instruments,Columbia, Md.), under-laid with a solution of 20% (wt/wt) sucrose, andsubjected to 2 hours centrifugation at 28,000 rpm in a Beckman SW28rotor. The resulting pellet is suspended in PBS and layered onto a20-60% (wt/wt) sucrose gradient and subjected to 2 hours centrifugationat 40,000 rpm in a Beckman SW41ti rotor.

The gradient is then fractionated into approximately 10×1 ml aliquots(starting at the top, 20%-end, of the gradient). Samples are taken fromfractions 1-9 and are electrophoresed on 8-16% SDS polyacrylamide gels.The supernatants from 293/synthetic Pol, Env or Gag cells give muchstronger bands than supernatants from 293/native Pol, Env or Gag cells.

Example 4 In Vivo Immunogenicity of Synthetic Pol, Gag and EnvExpression Cassettes

A. Immunization

To evaluate the possibly improved immunogenicity of the synthetic Pol,Gag and Env expression cassettes, a mouse study is performed. Theplasmid DNA, pCMVKM2 carrying the synthetic Gag expression cassette, isdiluted to the following final concentrations in a total injectionvolume of 100 μl: 20 μg, 2 μg, 0.2 μg, 0.02 and 0.002 μg. To overcomepossible negative dilution effects of the diluted DNA, the total DNAconcentration in each sample is brought up to 20 μg using the vector(pCMVKM2) alone. As a control, plasmid DNA of the native Gag expressioncassette is handled in the same manner. Twelve groups of four to tenBalb/c mice (Charles River, Boston, Mass.) are intramuscularly immunized(50 μl per leg, intramuscular injection into the tibialis anterior)according to the schedule in Table 1.

TABLE 1 Gag or Env Concentration of Gag or Immunized at Group ExpressionCassette Env plasmid DNA (μg) time (weeks): 1 Synthetic 20 0¹, 4 2Synthetic 2 0, 4 3 Synthetic 0.2 0, 4 4 Synthetic 0.02 0, 4 5 Synthetic0.002 0, 4 6 Synthetic 20 0 7 Synthetic 2 0 8 Synthetic 0.2 0 9Synthetic 0.02 0 10 Synthetic 0.002 0 11 Native 20 0, 4 12 Native 2 0, 413 Native 0.2 0, 4 14 Native 0.02 0, 4 15 Native 0.002 0, 4 16 Native 200 17 Native 2 0 18 Native 0.2 0 19 Native 0.02 0 20 Native 0.002 0 ¹=initial immunization at “week 0”

Groups 1-5 and 11-15 are bled at week 0 (before immunization), week 4,week 6, week 8, and week 12. Groups 6-20 and 16-20 are bled at week 0(before immunization) and at week 4.

B. Humoral Immune Response

The humoral immune response is checked with an anti-HIV Pol, Gag or Envantibody ELISAs (enzyme-linked immunosorbent assays) of the mice sera 0and 4 weeks post immunization (groups 5-12) and, in addition, 6 and 8weeks post immunization, respectively, 2 and 4 weeks post secondimmunization (groups 1-4).

The antibody titers of the sera are determined by anti-Pol, anti-Gag oranti-Env antibody ELISA. Briefly, sera from immunized mice are screenedfor antibodies directed against the HIV p55 Gag protein, an Env protein,e.g., gp160 or gp120 or a Pol protein, e.g., p6, prot or RT. ELISAmicrotiter plates are coated with 0.2 μg of Pol, Gag or Env protein perwell overnight and washed four times; subsequently, blocking is donewith PBS-0.2% Tween (Sigma) for 2 hours. After removal of the blockingsolution, 100 μl of diluted mouse serum is added. Sera are tested at1/25 dilutions and by serial 3-fold dilutions, thereafter. Microtiterplates are washed four times and incubated with a secondary,peroxidase-coupled anti-mouse IgG antibody (Pierce, Rockford, Ill.).ELISA plates are washed and 100 μl of 3, 3′, 5, 5′-tetramethyl benzidine(TMB; Pierce) is added per well. The optical density of each well ismeasured after 15 minutes. The titers reported are the reciprocal of thedilution of serum that gave a half-maximum optical density (O.D.).

Synthetic expression cassettes will provide a clear improvement ofimmunogenicity relative to the native expression cassettes.

C. Cellular Immune Response

The frequency of specific cytotoxic T-lymphocytes (CTL) is evaluated bya standard chromium release assay of peptide pulsed mouse (Balb/c, CB6F1and/or C3H) CD4 cells. Pol, Gag or Env expressing vaccinia virusinfected CD-8 cells are used as a positive control. Briefly, spleencells (Effector cells, E) are obtained from the mice immunized asdescribed above are cultured, restimulated, and assayed for CTL activityagainst Gag peptide-pulsed target cells as described (Doe, B., andWalker, C. M., AIDS 10(7):793-794, 1996). Cytotoxic activity is measuredin a standard ⁵¹Cr release assay. Target (T) cells are cultured witheffector (E) cells at various E:T ratios for 4 hours and the average cpmfrom duplicate wells are used to calculate percent specific ⁵¹Crrelease.

Cytotoxic T-cell (CTL) activity is measured in splenocytes recoveredfrom the mice immunized with HIV Gag or Env DNA. Effector cells from theGag or Env DNA-immunized animals exhibit specific lysis of Pol, Gag orEnv peptide-pulsed SV-BALB (MHC matched) targets cells, indicative of aCTL response. Target cells that are peptide-pulsed and derived from anMHC-unmatched mouse strain (MC57) are not lysed.

Thus, synthetic Pol, Env and Gag expression cassettes exhibit increasedpotency for induction of cytotoxic T-lymphocyte (CTL) responses by DNAimmunization.

Example 5 DNA-immunization of Non-Human Primates Using a Synthetic Pol,Env or Gag Expression Cassette

Non-human primates are immunized multiple times (e.g., weeks 0, 4, 8 and24) intradermally, mucosally or bilaterally, intramuscular, into thequadriceps using various doses (e.g., 1-5 mg) synthetic Pol, Gag- and/orEnv-containing plasmids. The animals are bled two weeks after eachimmunization and ELISA is performed with isolated plasma. The ELISA isperformed essentially as described in Example 4 except the secondantibody-conjugate is an anti-human IgG, g-chain specific, peroxidaseconjugate (Sigma Chemical Co., St. Louis, Md. 63178) used at a dilutionof 1:500. Fifty μg/ml yeast extract is added to the dilutions of plasmasamples and antibody conjugate to reduce non-specific background due topreexisting yeast antibodies in the non-human primates.

Further, lymphoproliferative responses to antigen can also be evaluatedpost-immunization, indicative of induction of T-helper cell functions.

Synthetic Pol, Env and Gag plasmid DNA are expected to be immunogenic innon-human primates.

Example 6 In Vitro Expression of Recombinant Sindbis RNA and DNAContaining the Synthetic Pol, Env and Gag Expression Cassette

To evaluate the expression efficiency of the synthetic Pol, Env and Gagexpression cassette in Alphavirus vectors, the selected syntheticexpression cassette is subcloned into both plasmid DNA-based andrecombinant vector particle-based Sindbis virus vectors. Specifically, acDNA vector construct for in vitro transcription of Sindbis virus RNAvector replicons (pRSIN-luc; Dubensky, et al., J. Virol. 70:508-519,1996) is modified to contain a Pmel site for plasmid linearization and apolylinker for insertion of heterologous genes. A polylinker isgenerated using two oligonucleotides that contain the sites XhoI, Pmll,ApaI, NarI, XbaI, and NotI (XPANXNF, and XPANXNR).

The plasmid pRSIN-luc (Dubensky et al., supra) is digested with XhoI andNotI to remove the luciferase gene insert, blunt-ended using Klenow anddNTPs, and purified from an agarose get using GeneCleanII (Bio101,Vista, Calif.). The oligonucleotides are annealed to each other andligated into the plasmid. The resulting construct is digested with NotIand SacI to remove the minimal Sindbis 3′-end sequence and A₄₀ tract,and ligated with an approximately 0.4 kbp fragment from PKSSIN1-BV (WO97/38087). This 0.4 kbp fragment is obtained by digestion of pKSSIN1-BVwith NotI and Sad, and purification after size fractionation from anagarose gel. The fragment contains the complete Sindbis virus 3′-end, anA₄₀ tract and a Pmel site for linearization. This new vector constructis designated SINBVE.

The synthetic HIV Pol, Gag and Env coding sequences are obtained fromthe parental plasmid by digestion with EcoRI, blunt-ending with Klenowand dNTPs, purification with GeneCleanII, digestion with SalI, sizefractionation on an agarose gel, and purification from the agarose gelusing GeneCleanII. The synthetic Pol, Gag or Env coding fragment isligated into the SINBVE vector that is digested with XhoI and PmtI. Theresulting vector is purified using GeneCleanII and is designatedSINBVGag. Vector RNA replicons may be transcribed in vitro (Dubensky etal., supra) from SINBVGag and used directly for transfection of cells.Alternatively, the replicons may be packaged into recombinant vectorparticles by co-transfection with defective helper RNAs or using analphavirus packaging cell line.

The DNA-based Sindbis virus vector pDCMVSIN-beta-gal (Dubensky, et al.,J. Virol. 70:508-519, 1996) is digested with SalI and XbaI, to removethe beta-galactosidase gene insert, and purified using GeneCleanII afteragarose gel size fractionation. The HIV Gag or Env gene is inserted intothe pDCMVSIN-beta-gal by digestion of SINBVGag with SalI and XhoI,purification using GeneCleanII of the Gag-containing fragment afteragarose gel size fractionation, and ligation. The resulting construct isdesignated pDSIN-Gag, and may be used directly for in vivoadministration or formulated using any of the methods described herein.

BHK and 293 cells are transfected with recombinant Sindbis RNA and DNA,respectively. The supernatants and cell lysates are tested with theCoulter capture ELISA (Example 2).

BHK cells are transfected by electroporation with recombinant SindbisRNA.

293 cells are transfected using LT-1 (Example 2) with recombinantSindbis DNA. Synthetic Gag- and/or Env-containing plasmids are used aspositive controls. Supernatants and lysates are collected 48 h posttransfection.

Pol, Gag and Env proteins can be efficiently expressed from both DNA andRNA-based Sindbis vector systems using the synthetic expressioncassettes.

Example 7 In Vivo Immunogenicity of Recombinant Sindbis Replicon VectorsContaining Synthetic Pol, Gag and/or Env Expression Cassettes

A. Immunization

To evaluate the immunogenicity of recombinant synthetic Pol, Gag and Envexpression cassettes in Sindbis replicons, a mouse study is performed.The Sindbis virus DNA vector carrying the synthetic Pol, Gag and/or Envexpression cassette (Example 6), is diluted to the following finalconcentrations in a total injection volume of 100 μl: 20 μg, 2 μg, 0.2μg, 0.02 and 0.002 μg. To overcome possible negative dilution effects ofthe diluted DNA, the total DNA concentration in each sample is broughtup to 20 μg using the Sindbis replicon vector DNA alone. Twelve groupsof four to ten Balb/c mice (Charles River, Boston, Mass.) areintramuscularly immunized (50 μl per leg, intramuscular injection intothe tibialis anterior) according to the schedule in Table 2.Alternatively, Sindbis viral particles are prepared at the followingdoses: 10³ pfu, 10⁵ pfu and 10′ pfu in 100 as shown in Table 3. SindbisPol, Env or Gag particle preparations are administered to mice usingintramuscular and subcutaneous routes (50 μl per site).

TABLE 2 Gag or Env Concentration of Gag Immunized at time GroupExpression Cassette or Env DNA (μg) (weeks): 1 Synthetic 20 0¹, 4 2Synthetic 2 0, 4 3 Synthetic 0.2 0, 4 4 Synthetic 0.02 0, 4 5 Synthetic0.002 0, 4 6 Synthetic 20 0 7 Synthetic 2 0 8 Synthetic 0.2 0 9Synthetic 0.02 0 10 Synthetic 0.002 0 ¹= initial immunization at “week0”

TABLE 3 Concentration of viral Immunized at time Group Gag or Envsequence particle (pfu) (weeks): 1 Synthetic 10³ 0¹, 4 2 Synthetic 10⁵0, 4 3 Synthetic 10⁷ 0, 4 8 Synthetic 10³ 0 9 Synthetic 10⁵ 0 10Synthetic 10⁷ 0 ¹= initial immunization at “week 0”

Groups are bled and assessment of both humoral and cellular (e.g.,frequency of specific CTLs) is performed, essentially as described inExample 4.

Example 8 Identification and Sequencing of a Novel HIV Type C Variants

A full-length clone, called 8_(—)5_ZA, encoding an HIV Type C wasisolated and sequenced. Briefly, genomic DNA from HIV-1 subtype Cinfected South African patients was isolated from PBMC (peripheral bloodmononuclear cells) by alkaline lysis and anion-exchange columns(Quiagen). To get the genome of full-length clones two halves wereamplified, that could later be joined together in frame within the Polregion using an unique Sal 1 site in both fragments. For theamplification, 200-800 ng of genomic DNA were added to the buffer andenzyme mix of the Expand Long Template PCR System after the protocol ofthe manufacturer (Boehringer Mannheim). The primer were designed afteralignments of known full length sequences. For the 5′ half a primer mixof 2 forward primers containing either thymidine (S1FCSacTA5′-GTTTCTTGAGCTCTGGAAGGGTTAATTTAC TCCAAGAA-3′, SEQ ID NO:38) or cytosineon position 20 (S1FTSacTA 5′-GTTTCTTGAGCTCTGGAAGGGTTAATTTACTCTAAGAA, SEQID NO:39) plus Sal 1 site, were used. The reverse primer were also a mixof two primers with either thymidine or cytosine on position 13(S145RTSalTA 5′-GTTTCTTGTCGACTTGTCCATGTATGGCTTCCCC T-3′, SEQ ID NO:40and S145RCSalTA 5′-GTTTCTTGTCGACTTGTCCATGCATGGCTTCCCT-3′ SEQ ID NO:41)and contained a Sal 1 site. The forward primer for the 3′ half was alsoa mixture of two primers (S245FASa1TA5′-GTTTCTTGTCGACTGTAGTCCAGGaATATGGCAAT TAG-3′ SEQ ID NO:42 andS245FGSalTA 5′-GTTTCTTGTCGACTGTAGTCCAGGgATATG GCAA TTAG-3′ SEQ ID NO:43)with Sal 1 site and adenine or guanine on position 12. The reverseprimer had a Not 1 site (S2_FullNotTA 5′-GTTTCTTGCGGCCGCTGCTAGAGATTTTCCACACTACCA-3′ SEQ ID NO:44). After amplification the PCR productswere purified using a 1% agarose gel and cloned into the pCR-XL-TOPOvector via TA cloning (Invitrogen). Colonies were checked by restrictionanalysis and sequence verified. For the full length sequence thesequences of the 5′- and 3′ half were combined. The sequence is shown inSEQ ID NO:33. Furthermore, important domains are shown in Table A.

Another clone, designated 12_(—)5/1ZA was also sequenced and is shown inSEQ ID NO:45.

As described in Example 1, synthetic expression cassettes are generatedusing one or more polynucleotide sequence obtained from 8_(—)5_ZA or12_(—)5/1ZA.

Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1. An expression cassette, comprising a polynucleotide sequence operablylinked to a promoter, wherein the polynucleotide sequence has at least90% sequence identity to SEQ ID NO:30; SEQ ID NO:31; or SEQ ID NO:32. 2.The expression cassette of claim 1, further comprising one or morenucleic acids encoding one or more viral polypeptides or antigens. 3.The expression cassette of claim 2, wherein the viral polypeptides orantigens are selected from the group consisting of Gag, Env, vif, vpr,tat, rev, vpu, nef and combinations thereof.
 4. The expression cassetteof claim 1, further comprising one or more nucleic acids encoding one ormore cytokines.
 5. A recombinant expression system for use in a selectedhost cell, comprising, the expression cassette of claim 1, and whereinsaid polynucleotide sequence is operably linked to control elementscompatible with expression in the selected host cell.
 6. The recombinantexpression system of claim 5, wherein said control elements are selectedfrom the group consisting of a transcription promoter, a transcriptionenhancer element, a transcription termination signal, polyadenylationsequences, sequences for optimization of initiation of translation, andtranslation termination sequences.
 7. The recombinant expression systemof claim 6, wherein said transcription promoter is selected from thegroup consisting of CMV, CMV+intron A, SV40, RSV, HIV-Ltr, MMLV-ltr, andmetallothionein.
 8. A cell comprising the expression cassette of claim1, and wherein said polynucleotide sequence is operably linked tocontrol elements compatible with expression in the selected cell.
 9. Thecell of claim 8, wherein the cell is a mammalian cell.
 10. The cell ofclaim 9, wherein the cell is selected from the group consisting of BHK,VERO, HT1080, 293, RD, COS-7, and CHO cells.
 11. The cell of claim 10,wherein said cell is a CHO cell.
 12. The cell of claim 8, wherein thecell is an insect cell.
 13. The cell of claim 12, wherein the cell iseither Trichoplusia ni (Tn5) or Sf9 insect cells.
 14. The cell of claim8, wherein the cell is a bacterial cell.
 15. The cell of claim 8,wherein the cell is a yeast cell.
 16. The cell of claim 8, wherein thecell is a plant cell.
 17. The cell of claim 8, wherein the cell is anantigen presenting cell.
 18. The cell of claim 17, wherein the antigenpresenting cell is a lymphoid cell selected from the group consisting ofmacrophage, monocytes, dendritic cells, B-cells, T-cells, stem cells,and progenitor cells thereof.
 19. The cell of claim 8, wherein the cellis a primary cell.
 20. The cell of claim 8, wherein the cell is animmortalized cell.
 21. The cell of claim 8, wherein the cell is a tumorcell.
 22. A composition for generating an immunological response,comprising the expression cassette of claim
 1. 23. The composition ofclaim 22, further comprising one or more Pol polypeptides.
 24. Thecomposition of claim 23, further comprising an adjuvant.
 25. Acomposition for generating an immunological response, comprising theexpression cassette of claim
 2. 26. The composition of claim 25, furthercomprising a Pol polypeptide.
 27. The composition of claim 26, furthercomprising a polypeptide encoded by a polynucleotide sequence operablylinked to a promoter, wherein the polynucleotide sequence encodes an HIVPol polypeptide that elicits a Pol-specific immune response, and furtherwherein the polynucleotide sequence encoding said polypeptide comprisesa nucleotide sequence having at least 90% sequence identity to SEQ IDNO:30; SEQ ID NO:31; or SEQ ID NO:32.
 28. The composition of claim 27,further comprising an adjuvant.
 29. A method of generating an immuneresponse in a subject, comprising, introducing the composition of claim22 into said subject under conditions that are compatible withexpression of said expression cassette in said subject.
 30. The methodof claim 29, wherein said expression cassette is introduced using a genedelivery vector.
 31. The method of claim 30, wherein the gene deliveryvector is a non-viral vector.
 32. The method of claim 30, wherein saidgene delivery vector is a viral vector.
 33. The method of claim 32,wherein said gene delivery vector is a Sindbis virus derived vector. 34.The method of claim 32, wherein said gene delivery vector is aretroviral vector.
 35. The method of claim 32, wherein said genedelivery vector is a lentiviral vector.
 36. The method of claim 30,wherein said composition is delivered by using a particulate carrier.37. The method of claim 30, wherein said composition is coated on a goldor tungsten particle and said coated particle is delivered to saidsubject using a gene gun.
 38. The method of claim 30, wherein saidcomposition is encapsulated in a liposome preparation.
 39. The method ofany one of claims 30-38, wherein said subject is a mammal.
 40. Themethod of claim 39, wherein said mammal is a human.
 41. The method ofclaim 29, where the method further comprises administration of apolypeptide derived from an HIV.
 42. The method of claim 41, whereinadministration of the polypeptide to the subject is carried out beforeintroducing said expression cassette.
 43. The method of claim 41,wherein administration of the polypeptide to the subject is carried outconcurrently with introducing said expression cassette.
 44. The methodof claim 41, wherein administration of the polypeptide to the subject iscarried out after introducing said expression cassette.
 45. Theexpression cassette of claim 2, wherein the viral polypeptides orantigens are selected from the group consisting of polypeptides derivedfrom hepatitis B, hepatitis C and combinations thereof.
 46. Anexpression cassette comprising the polynucleotide sequence of SEQ ID NO:30, SEQ ID NO: 31 or SEQ ID NO:
 32. 47. The expression cassette of claim46 further comprising a nucleotide sequence encoding a viral polypeptideselected from the group consisting of Gag, Env, vif, vpr, tat, rev, vpu,nef, and combinations thereof.
 48. A composition for generating animmunological response in a mammal comprising the expression cassette ofclaim
 46. 49. A method of generating an immune response in a mammal, themethod comprising the step of intramuscularly administering theexpression cassette of claim 46 to said mammal.
 50. The expressioncassette of claim 1, comprising a nucleotide sequence encoding an HIV-1Pol polypeptide, wherein the catalytic center region of theReverse-Transcriptase is modified to become non-functional, and whereinsaid nucleotide sequence has at least 90% sequence identity to SEQ IDNO:31.
 51. The expression cassette of claim 1, comprising a nucleotidesequence encoding an HIV-1 Pol polypeptide, wherein the catalytic centerand the primer grip region of the Reverse-Transcriptase are modified tobecome non-functional, and wherein said nucleotide sequence has at least90% sequence identity to SEQ ID NO:32.